Method for pretreating specimen and method for assaying biological substance

ABSTRACT

This invention relates to an assay device and a method for pretreating a specimen containing a biologically-relevant substance and then assaying the biologically-relevant substance. Biologically-relevant substances used in the assay method include microorganisms, cells, viruses, nucleic acids, polysaccharides, proteins (including antigens, antibodies, chromoproteins, and enzymes), peptides, nucleic acids, and small molecules. This pretreatment method removes contaminants from the biologically-relevant substances using supports such as magnetic particles, gels, resins, membranes, and solid-phased reagents. Therefore, in the assay method the following steps are generally carried out (i) a pretreatment step of treating a specimen using one or more first supports and then one or more second supports, and (ii) the assay step carried out after the pretreatment step. In particular, the pretreatment method reduces false positives and false negatives in the assay.

This application is the National Stage of International Application No.PCT/JP2009/071678, filed in the Japanese Patent Office on Dec. 25, 2009.International Application PCT/JP2009/071678 in turn claims priorityunder 35 USC §119(a)-(d) of Japanese Application No. 2009-175584, filedin the Japanese Patent Office on Jul. 28, 2009 and of JapaneseApplication No. 2008-31219, filed in the Japanese Patent Office on Dec.25, 2008.

TECHNICAL FIELD

The present invention relates to a method for pretreating a specimencontaining a biologically-relevant substance before being subjected toan assay and a system for assaying the biologically-relevant substancein the specimen.

BACKGROUND ART

Since the method of preparing a monoclonal antibody was established, asa method for assaying a biologically-relevant substance of interest in aspecimen, an immunoassay such as enzyme immunoassay has been employed asthe main assay method. When using such an immunoassay, it is possible toperform direct assay because of its high specificity, and assay can beperformed with high sensitivity. Further, recently, regarding theseassay methods, steps from the step after setting a collected specimen tothe step of obtaining assay results have been automated by the assaysystem. Further, in order to accelerate assay more, reagents in whichconcentrations of a solid-phased antibody and a conjugate are higherthan ever before have been developed. However, when using reagents inmore concentrated form, nonspecific reactions, which are conventionallyunrelated at the time of assay, are often caused.

It is considered that causes of nonspecific reactions are the variety oftarget substances, the presence of immune analogs, the variety ofantigens and the variety of antibodies. In the immunoassay system, assubstances causing various nonspecific reactions, IgA-type, IgM-type andIgG-type heterophilic antibodies (antibodies that react between animalsof different species: HAMA, anti-BSA antibody, etc.) and biologicalcomponents (e.g., rheumatoid factor, cryoglobulin and M protein) arepresent, and there is a case where they show false positive inimmunoassay as a result of a nonspecific reaction (see Non-patentdocuments 1, 2 and 3). Moreover, since cancer-associated antigens suchas sugar chain exist on the surfaces of bacteria, in the case ofinfection caused by bacteria, false positive is often shown in cancertests without cancer. Furthermore, there is a case where false positiveis shown due to a nonspecific reaction caused, for example, by bacterialinfection at the time of surgery for removing an organ from a cancerpatient.

Further, a nonspecific reaction often occurs during pregnancy or whenbeing affected with liver disease, kidney disease or the like. Moreover,recombinant antigens are used for many recently-developed reagents. Itis known that due to the presence of bacterial components used at thetime of preparation of these recombinant antigens, antibodies againstthese bacteria affect the assay system. In general, when performingimmunoassay, inhibitors against these nonspecific reactive substancesare added. However, since there is a limitation on the adding amountsfor reagents, sufficient inhibitory effects are not necessarilyobtained. Therefore, it is difficult for currently-used assay systems toremove nonspecific reaction factors and to sufficiently inhibitnonspecific reactions to perform assay.

PRIOR ART DOCUMENTS

-   [Non-patent document 1] Marlen Bouillon, et al., Reduced frequency    of blood donors with false-positive HIV-1 and -2 antibody EIA    reactivity after elusion of low-affinity nonspecific natural    antibodies, TRANSFUSION, Volume 42, August 2002, 1046-1052-   [Non-patent document 2] Johan Bjerner, et al., Incidence and    Prevention, Clinical Chemistry, 48:4 613-621 (2002)-   [Non-patent document 3] Michael Covinsky, et al., An IgM λ, Antibody    to Escherichia coli Produces False-Positive Results in Multiple    Immunometric Assays Michael Covinsky, Clinical Chemistry, 46:8    1157-1161 (2000)

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

The present invention was made in consideration of the above-describedcircumstances. The purpose of the present invention is to provide apretreatment method in which a contaminant can be removed in advancefrom a specimen to be subjected to an assay and an assay method usingthe pretreated specimen.

Means for Solving the Problems

The present inventor diligently made researches in order to solve theabove-described problems and found that a biologically-relevantsubstance can be highly sensitively assayed when the assay is carriedout after pretreating a specimen. Thus the present invention wasachieved. That is, the present invention relates to a system forassaying a biologically-relevant substance in a specimen, comprising:

a first support to which a substance having affinity to a contaminantcontained in the specimen, a substance that inactivates the contaminant,or a substance having affinity to the biologically-relevant substance inthe specimen is immobilized; and

a second support selected from a support to which a reagent fordetecting the biologically-relevant substance is immobilized and asupport made of a solid-phased reagent for detecting thebiologically-relevant substance.

In the present invention, the biologically-relevant substance may be anantigen or antibody or a nucleic acid.

Further, the contaminant in the specimen may be a nonspecific reactionfactor. The nonspecific reaction factor may be at least one selectedfrom the group consisting of an immunoglobulin, an M protein, aheterophilic antibody and a rheumatoid factor.

The first support is, for example, at least one selected from the groupconsisting of magnetic particles, a gel, a resin and a membrane.Further, the magnetic particles are preferably capable of being held ina dispensing chip attached to a dispensing nozzle. In this case, atreatment such as separation, washing and suspension is preferablycarried out in the dispensing chip using the magnetic particles.

Further, the reagent for detecting the biologically-relevant substancepreferably comprises a labeled antigen or a labeled antibody against thebiologically-relevant substance or a primer and a probe for amplifyingthe biologically-relevant substance.

In one embodiment of the present invention, solid-phasing of the reagentfor detecting is preferably carried out by freeze-drying(lyophilization).

Further, the specimen, the first support and the second support arepreferably held in different holding portions such as wells and chipsrespectively.

Moreover, the system of the present invention is characterized in that aholding portion (well or chip) in which the specimen is held, a holdingportion in which the first support is held and a holding portion inwhich the second support is held are arranged in an approximate straightline.

Further, the system of the present invention is characterized in thatthe specimen holding portion in which the specimen is held, the holdingportion in which the first support is held and the holding portion inwhich the second support is held are integrated into a cartridge.

In the above-described system, the holding portions are preferablysealable.

Moreover, the cartridge of the present invention is characterized inthat it comprises: a holding portion, in which a first support to whicha substance having affinity to a contaminant contained in a specimen, asubstance that inactivates the contaminant, or a substance havingaffinity to the biologically-relevant substance in the specimen isimmobilized is held in advance; a holding portion, in which a secondsupport selected from a support to which a reagent for detecting thebiologically-relevant substance is immobilized and a support made of asolid-phased reagent for detecting the biologically-relevant substanceis held; and a specimen holding portion in which the specimen is held.

In the above-described cartridge, the first support is preferablymagnetic particles.

Further, in this cartridge, solid-phasing is preferably carried out byfreeze-drying.

In this cartridge, the holding portions may be sealable.

Moreover, this cartridge is characterized in that the specimen holdingportion in which the specimen is held, the holding portion in which thefirst support is held and the holding portion in which the secondsupport is held are arranged in an approximate straight line.

The pretreatment method of the present invention is a method forpretreating a specimen before assaying a biologically-relevant substancein the specimen, comprising a step of treating the specimen using afirst support to which a substance having affinity to a contaminantcontained in the specimen, a substance that inactivates the contaminant,or a substance having affinity to the biologically-relevant substance inthe specimen is immobilized.

Moreover, the pretreatment method of the present invention ischaracterized in that it further comprises a step of preparing a secondsupport selected from a support to which a reagent for detecting thebiologically-relevant substance is immobilized and a support made of asolid-phased reagent for detecting the biologically-relevant substance.

In the above-described pretreatment method, the biologically-relevantsubstance may be an antigen or antibody or a nucleic acid.

Examples of the contaminant include a nonspecific reaction factor.

Further, the nonspecific reaction factor may be at least one selectedfrom the group consisting of an immunoglobulin, an M protein, aheterophilic antibody and a rheumatoid factor.

The first support is, for example, at least one selected from the groupconsisting of magnetic particles, a gel, a resin and a membrane.

Further, the reagent for detecting the biologically-relevant substancepreferably comprises a labeled antigen or a labeled antibody against thebiologically-relevant substance or a primer and a probe for amplifyingthe biologically-relevant substance.

Further, in the pretreatment method, solid-phasing is preferably carriedout by freeze-drying.

Moreover, in the present invention, the pretreated specimen may beprovided to an assay device to assay the biologically-relevant substancein the specimen.

Further, the assay is preferably an immunoassay or an assay by means ofa nucleic acid amplification method.

Examples of the nucleic acid amplification method include a PCR methodand an isothermal amplification method. At the time of amplification ofa nucleic acid, a mixture of a target nucleic acid-containing solutionand an amplification reagent is preferably held in a holding portionsuch as a well and a chip and sealed with a hydrophobic fluid such as amineral oil.

In the present invention, the pretreatment of the specimen and the assayof the treated specimen can be carried out successively.

Moreover, the nucleic acid amplification device of the present inventionis characterized in that it comprises:

(a) a specimen holding portion in which a specimen is held;

(b) a first holding portion in which trapping particles for trapping atarget nucleic acid from the specimen are held;

(c) a second holding portion in which a reagent for detecting the targetnucleic acid is held;

(d) a dispensing mechanism for dispensing the specimen into the specimenholding portion, a mechanism for mixing the specimen with the trappingparticles to extract the target nucleic acid from the specimen, and amechanism for mixing the extracted target nucleic acid with the reagentfor detecting; and(e) a mechanism selected from the group consisting of: a mechanism forpouring a hydrophobic fluid, which has a specific gravity smaller thanthat of a mixed fluid of the target nucleic acid and the reagent fordetecting, into the second holding portion; a mechanism for removing orputting lids for covering the respective holding portions; a mechanismfor irradiating an irradiating light for letting the target nucleic acidfluoresce; a mechanism for receiving a light from the target nucleicacid irradiated with the irradiating light to detect the target nucleicacid; and a mechanism in which the mechanisms are combined.

Advantageous Effect of the Invention

According to the present invention, a biologically-relevant substancecan be assayed with significantly high sensitivity.

In the conventional systems for assaying a specimen, abiologically-relevant substance is assayed with a contaminant being notsufficiently removed from the specimen. According to the presentinvention, the contaminant can be removed and it is possible to increasesensitivity at the time of assaying the biologically-relevant substance.Further, according to the present invention, the pretreatment of thespecimen and the assay of the biologically-relevant substance in thepretreated specimen can be carried out automatically and successively.Moreover, in the conventional systems, it is required to manually add areagent that deactivates a contaminant to a specimen to be assayed inorder to avoid intervention of the contaminant, but according to thepresent invention, it is no longer necessary to manually add such areagent. Therefore, assay results can be very conveniently obtained.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an explanatory drawing exemplifying combinations of a firstsupport and a second support.

FIG. 2 is an explanatory drawing for schematically explaining anembodiment in which a contaminant is removed using a first support towhich a substance having affinity to the contaminant is immobilized.

FIG. 3 is an explanatory drawing for schematically showing an embodimentin which a contaminant is decomposed using a substance that decomposesthe contaminant.

FIG. 4 is an explanatory drawing for schematically explaining anembodiment in which a biologically-relevant substance is extracted usinga first support to which a substance having affinity to thebiologically-relevant substance is immobilized.

FIG. 5 is an explanatory drawing for schematically explaining anembodiment in which a biologically-relevant substance is extracted andlabeled using a second support to which a substance having affinity tothe biologically-relevant substance is immobilized.

FIG. 6 is an explanatory drawing for schematically showing all the stepsfrom pretreatment using magnetic particles to which an antibody againsta nonspecific reaction factor is immobilized to detection of an antigencontained in a specimen.

FIG. 7 is an explanatory drawing for schematically showing all the stepsfrom pretreatment using a support to which an antibody against anonspecific reaction factor is immobilized to detection of an antigencontained in a specimen.

FIG. 8 is an explanatory drawing for explaining a treatment of removinga contaminant using magnetic particles to which an antibody against anonspecific reaction factor is immobilized.

FIG. 9 is a flow chart of a treatment of removing a contaminant usingmagnetic particles to which an antibody against a nonspecific reactionfactor is immobilized.

FIG. 10 is an explanatory drawing for explaining a treatment of removinga contaminant using a column to which an antibody against a nonspecificreaction factor is immobilized.

FIG. 11 is a flow chart of a treatment of removing a contaminant using acolumn to which an antibody against a nonspecific reaction factor isimmobilized.

FIG. 12 is an explanatory drawing for explaining a treatment of removinga contaminant using a membrane to which an antibody against anonspecific reaction factor is immobilized.

FIG. 13 is a flow chart of a treatment of removing a contaminant using amembrane to which an antibody against a nonspecific reaction factor isimmobilized.

FIG. 14 is an explanatory drawing for explaining a treatment of removinga contaminant using a gel to which a reducing agent is immobilized.

FIG. 15 is a flow chart of a treatment of removing a contaminant using agel to which a reducing agent is immobilized.

FIG. 16 is an explanatory drawing for explaining an extraction treatmentusing magnetic particles to which a probe having affinity to nucleicacid is immobilized.

FIG. 17 is a flow chart of a treatment of extracting a nucleic acidusing magnetic particles to which a probe having affinity to the nucleicacid is immobilized.

FIG. 18 is an explanatory drawing for schematically explaining amagnetic particle to which an antibody against an antigen isimmobilized, a plate to which the antibody against the antigen isimmobilized, and a bead to which the antibody against the antigen isimmobilized.

FIG. 19 is an explanatory drawing for schematically explaining anembodiment in which an antigen is detected by trapping and labeling theantigen using a second support.

FIG. 20 is an explanatory drawing for explaining an immunoassay usingmagnetic particles.

FIG. 21 is an explanatory drawing for explaining an immunoassay using anantigen separation/immobilization tube.

FIG. 22 is a block diagram showing the functions of the assay system.

FIG. 23 is a flow chart of a case where a pretreatment is carried outusing magnetic particles to which an antibody against a nonspecificreaction factor is immobilized and subsequently an immunoassay iscarried out.

FIG. 24 is a flow chart of a case where a pretreatment is carried outusing a column to which an antibody against a nonspecific reactionfactor is immobilized and subsequently an immunoassay is carried out.

FIG. 25 is a schematic view of an assay system utilizing a cartridge inwhich magnetic particles for pretreatment and a substrate solution areheld in advance.

FIG. 26 is an explanatory drawing for schematically explaining a mode ofoperation of the assay system utilizing the cartridge.

FIG. 27 is an explanatory drawing for showing the pretreatment step inthe assay system utilizing the cartridge.

FIG. 28 is an explanatory drawing for showing the assay step utilizingthe cartridge.

FIG. 29 is an explanatory drawing for schematically explaining acartridge having another form.

FIG. 30 is an explanatory drawing for schematically showing thepretreatment step from extraction of a nucleic acid from a specimen topreparation of a second support.

FIG. 31 is an explanatory drawing for schematically explaining atreatment portion in which 12 lines of a plurality of wells are arrangedin lines.

FIG. 32 is a block diagram showing the functions of the assay system.

FIG. 33 is a flow chart showing the treatment process carried out by theassay system.

FIG. 34 is a schematic view of an assay system in which a nucleic acidcan be pretreated utilizing a cartridge.

FIG. 35 is a perspective cross portional view of a cartridge in whichwells holding a master mixture are partially taken along thelongitudinal direction of the cartridge.

FIG. 36 is an explanatory drawing for explaining an embodiment in whichwells holding an assay sample and wells holding a master mixture areseparated from a cartridge body.

FIG. 37 is an explanatory drawing for explaining an embodiment in whichdetection of a nucleic acid is carried out using a plurality of wellsarranged in a line.

FIG. 38 is a perspective view showing a cartridge having a plurality oftreatment lines in which wells are arranged and nozzle units which moveon this cartridge along the treatment lines.

FIG. 39 is a perspective view of the tip portion of a nozzle unit havinga pumping opening, an optical fiber for sending a trigger light and alens for detection.

FIG. 40 is a cross portional view of the tip portion of a nozzle unittaken along a plane parallel to the drawing direction of an opticalfiber.

FIG. 41 is a functional block diagram showing the functions of a nucleicacid detection apparatus.

FIG. 42 is a perspective view schematically showing a nucleic aciddetection apparatus having only one nozzle unit having a pumpingopening, an optical fiber for sending a trigger light and a lens fordetection.

FIG. 43 is a perspective view showing the main portion of a nucleic aciddetection apparatus having nozzles for dispensing provided to respectivetreatment lines and a single nucleic acid detector separately.

FIG. 44 is a perspective view of a nucleic acid detection apparatushaving nucleic acid detectors corresponding to respective treatmentlines.

FIG. 45 is a cross portional view showing an embodiment in which anoptical fiber is provided to the outside of a nozzle for dispensing.

FIG. 46 is an explanatory drawing schematically showing a nucleic aciddetection apparatus having a single detector and a switching apparatusfor allowing the detector to selectively correspond to each of wells fordetection.

FIG. 47 is a graph showing a standard curve for obtaining an AFP valuebased on addition of AFP to serum.

FIG. 48 is a graph showing a standard curve for obtaining an AFP valuebased on addition of AFP to PBS buffer solution.

BEST MODE FOR CARRYING OUT THE INVENTION 1. Summary

The present invention relates to a system for assaying a specimenutilizing a plurality of types of supports having different functionsand a method for pretreating the specimen for the purpose of assaying atarget substance in the specimen. The assay system of the presentinvention can be used for a specimen containing a biologically-relevantsubstance. The specimen is treated with a plurality of supports havingdifferent functions and the biologically-relevant substance as a targetis assayed with high accuracy. As the plurality of supports havingdifferent functions, for example, it is possible to use: a first supportto which a substance having affinity to a contaminant contained in thespecimen, a substance that inactivates the contaminant, or a substancehaving affinity to the biologically-relevant substance in the specimenis immobilized; and a second support selected from a support to which areagent for detecting the biologically-relevant substance is immobilizedand a support made of a solid-phased reagent for detecting thebiologically-relevant substance. “Having affinity” means that substances(substances A and B) chemically or physically interact with each otherto enhance bonding thereof. Further, to “inactivate” means thatpossessed functions are inhibited. As combinations of the substance Aand the substance B having affinity to the substance A, for example, anantigen and an antibody, a ligand and a receptor, a nucleic acid and acomplementary strand thereof, etc. are exemplified. Examples of thefirst support for removing the contaminant include a support having asubstance (e.g., magnetic particles, a column, a filtering material, apolymeric material, etc.) to which an antibody against a nonspecificreaction factor is immobilized. Further, examples of the first supportfor decomposing the contaminant include a support having a reducingagent for decomposing the nonspecific reaction factor. Further, examplesof the first support for extracting the biologically-relevant substanceinclude magnetic particles to which a probe having affinity to a nucleicacid as the biologically-relevant substance is immobilized. By utilizingthese exemplified first supports, removal of the contaminant from thespecimen and extract of the biologically-relevant substance from thespecimen can be carried out.

As shown in FIG. 1, as the first support, the following three types ofsupports are exemplified: a support to which a substance having affinityto a contaminant contained in a specimen is immobilized; a support towhich a substance that inactivates the contaminant is immobilized; and asupport to which a substance having affinity to a biologically-relevantsubstance in the specimen is immobilized. As the second support, thefollowing two types of supports are exemplified: a support to which areagent for detecting the biologically-relevant substance isimmobilized; and a support made of a solid-phased reagent for detectingbiologically-relevant substance. Therefore, it is considered that thenumber of combinations of the first support and the second support is atleast 6. By treating the specimen using these combinations, varioustreatment embodiments can be formed.

FIGS. 2-5 are explanatory drawings for schematically explaining theprinciple of the system of the present invention. For example, as shownin FIG. 2, by treating a specimen using a support to which a substancehaving affinity to a contaminant contained in the specimen isimmobilized as a first support, the support traps the contaminant, andby collecting (removing) the support, the contaminant can be removedfrom the specimen. Further, as shown in FIG. 3, by utilizing a supportto which a substance that inactivates a contaminant contained in aspecimen (inactivating substance) is immobilized, as a first support,the contaminant in the specimen can be decomposed, and as a result, thecontaminant can be removed from the specimen. After obtaining abiologically-relevant substance, by using a support to which a reagentfor detecting the biologically-relevant substance is immobilized as asecond support, the biologically-relevant substance in the specimen canbe detected. Further, as shown in FIG. 4, by using a support to which asubstance having affinity to the biologically-relevant substance in thespecimen is immobilized as a first support, only thebiologically-relevant substance can be extracted from the specimen, andit is possible to prevent inhibition by the contaminant at the time ofassay of the biologically-relevant substance, etc.

In the present invention, as shown in FIG. 1, it is also possible toemploy combinations of a first support and a second support other thanthose exemplified above. Depending on respective combinations, differenttreatments can be applied to the specimen. Further, after using thefirst support to which the substance having affinity to the contaminantis immobilized, the specimen can be further treated using the firstsupport to which the substance having affinity to thebiologically-relevant substance is immobilized.

After obtaining the biologically-relevant substance using the firstsupport, the biologically-relevant substance can be assayed using thesecond support. The first support is a support for highly purifying orextracting the biologically-relevant substance in the specimen, and isused for the pretreatment of the present invention. The second supportis a support to which an assay reagent for detecting thebiologically-relevant substance is immobilized, or a support made of thesolid-phased reagent. In the present invention, an assay step can becarried out by separately performing a reaction with the reagent withoutpreparing the second support. However, in consideration of totalautomation using an apparatus, the second support is preferably preparedin advance. Further, by preparing not only the second support but alsothe first support in advance, more convenient operation with higherefficiency can be realized.

As shown in FIG. 5, the second support to which the substance havingaffinity to the biologically-relevant substance (affinity substance) isimmobilized is bound to the biologically-relevant substance, and thesecond support to which a labeling substance is immobilized is bound tothe biologically-relevant substance, thereby detecting thebiologically-relevant substance. When using the first support havingaffinity to the biologically-relevant substance, there is a case whereuse of the second support having affinity to the biologically-relevantsubstance can be omitted. In this case, for a specimen treated using thefirst support, the second support to which the labeling substance isimmobilized can be used immediately.

Further, the pretreatment method of the present invention is a methodfor pretreating a specimen before assaying a biologically-relevantsubstance in the specimen, characterized in that it comprises a step oftreating the specimen using a first support to which a substance havingaffinity to a contaminant contained in the specimen, a substance thatinactivates the contaminant, or a substance having affinity to thebiologically-relevant substance in the specimen is immobilized. Thepretreatment means both removing the contaminant contained in thespecimen and extracting or purifying the biologically-relevant substancecontained in the specimen.

The pretreatment of the present invention may further comprise a step ofpreparing a second support to which a substance having affinity to abiologically-relevant substance and/or a substance for labeling thebiologically-relevant substance is immobilized. The preparation of thesecond support can be carried out simultaneously with or before or afterthe treatment using the first support. When the biologically-relevantsubstance is an antigen or antibody, preparation for labeling anddetecting an antibody or antigen in a subsequent assay step iscompleted. When the biologically-relevant substance is a nucleic acid,preparation for labeling and detecting a nucleic acid in a subsequentstep is completed.

After carrying out the pretreatment described above, the assay step iscarried out. When the biologically-relevant substance as an assay targetis an antigen or antibody, examples of the first support include holdingbodies, such as magnetic particles, a gel and a membrane, to which asubstance having affinity to a contaminant in a specimen is immobilized,and examples of the second support include a substance (e.g., magneticparticles and beads) to which an antibody against the antigen isimmobilized.

Further, when the biologically-relevant substance is a nucleic acid suchas a DNA and RNA, examples of the first support include a support towhich a substance having affinity to a DNA/RNA in a specimen isimmobilized, and examples of the second support include a support madeof a solid-phased reaction reagent (e.g., probe, primer and mastermixture) required for amplifying and assaying a specific portion of anucleotide sequence of a DNA/RNA extracted, separated or purified.

More specifically, for example, when the biologically-relevant substanceis a tumor marker (e.g., CA19-9), in order to prevent a false-positivereaction at the time of assay, as a first support, a support (magneticparticles or a nonmagnetic solid) to which a substance that binds to acontaminant for removing a reaction-inhibiting substance such as IgM(e.g., IgM antibody) is immobilized is used. Further, as a secondsupport, a magnetic or nonmagnetic solid to which a tumor markerantibody (e.g., anti-CA19-9 antibody) is immobilized is used. The assayis performed by adding the biologically-relevant substance (CA19-9 inthe example above) treated using the first support and a substratesolution to a container containing the second support.

When the biologically-relevant substance as the assay target is anucleic acid (e.g., influenza virus RNA), as the first support, magneticparticles for extracting, separating or purifying a virus RNA are used,and as the second support, a support comprising a reaction reagentrequired for amplifying and assaying (e.g., PCR) a specific portion of anucleotide sequence of the above-described RNA extracted, separated orpurified is used. The assay is performed by adding thebiologically-relevant substance (influenza virus RNA in the exampleabove) treated using the first support and the reaction reagent foramplification to a container containing the second support.

In one embodiment of the present invention, the treatment for removingthe nonspecific reaction factor in the specimen and the treatment forpreparing the second support to which a substance having affinity to thebiologically-relevant substance and/or labeling thebiologically-relevant substance is immobilized are carried outsuccessively in one apparatus. An apparatus realizing this is called anassay system in the present invention.

As shown in FIG. 6, in the assay system, as a pretreatment step, a stepof treating a specimen containing a biologically-relevant substance,such as a step of removing a contaminant, and a step of preparing asecond support are carried out. A step of detecting thebiologically-relevant substance in the specimen has a labeling reactionstep for labeling the biologically-relevant substance in the specimenand an assay step for assaying the labeled biologically-relevantsubstance. Therefore, broadly speaking, in the assay system, thefollowing 3 steps are carried out: (i) a step of treating a specimenusing a first support; (ii) a step of preparing a second support: and(iii) an assay step carried out after a pretreatment. Basically, thestep (i) is called a pretreatment. But the pretreatment step may be acombination of the step (i) and the step (ii). It is understood that thestep (i) is a treatment for creating an environment in a test tube forhighly purifying or extracting a biologically-relevant substance as anassay target, and that the step (ii) is for creating an environment in atest tube containing a reagent for detecting the biologically-relevantsubstance. It means a kind of step for preparing a sample and a reagentfor assaying the sample for the purpose of allowing thebiologically-relevant substance in the specimen to be assayed. In FIG.6, preparation of the second support is included in the pretreatmentstep.

The step of treating the specimen using the first support means a stepof removing a contaminant and a step of highly purifying or extractingthe biologically-relevant substance. This step can be suitably selecteddepending on the preparation of the second support. For example,techniques such as: (a) a technique of selectively collecting a desiredbiologically-relevant substance utilizing magnetic particles; and (b) atechnique of simultaneously collecting a plurality of types ofbiologically-relevant substances desired can be employed. When abiologically-relevant substance as a detection target is a protein suchas an antigen and an antibody, mainly the following embodiments can beemployed: (1) a technique of trapping and removing a nonspecificreaction factor utilizing magnetic particles to which an antibodyagainst the nonspecific reaction factor is immobilized; (2) a techniqueof trapping and removing a nonspecific reaction factor utilizing anaffinity gel to which an antibody against the nonspecific reactionfactor is immobilized; (3) a technique of trapping and removing anonspecific reaction factor utilizing a filter to which an antibodyagainst the nonspecific reaction factor is immobilized; (4) a techniqueof trapping and removing a nonspecific reaction factor utilizing aplastic support to which an antibody against the nonspecific reactionfactor is immobilized; and (5) a technique of decomposing a nonspecificreaction factor using a gel to which a reducing agent is immobilized.When the biologically-relevant substance is a nucleic acid such as a DNAand RNA, for example, a technique of extracting a target nucleic acidusing magnetic particles to which a probe that can bind to the targetnucleic acid is immobilized can be employed.

In the assay system of the present invention, the pretreatment step iscarried out and then an assay such as an immunoassay and a nucleic acidassay can be carried out successively. The assay system has a nozzle, apipette chip as a dispensing chip, a holding portion such as a wellplate in which a plurality of wells are arranged, a pump mechanism, etc.In the holding portion, a solution of magnetic particles, a washingsolution, an enzyme-labeling solution, a substrate solution, etc. can beheld. The movement of the pipette chip can be automatically controlledby a motor, a motor controller, etc. The material of the well may besuitably selected in view of the detection method. For example, in thecase of performing CLIA or CLEIA, the well may be made of an opaquematerial which is not affected by mutual luminescence, and in the caseof performing EIA (ELISA), the well may be made of a transparentmaterial because a transmitted light is handled. As describedhereinbelow, magnetic particles for trapping a nonspecific reactionfactor mean, for example, a magnetic substance, which has a surface towhich an antibody against the nonspecific reaction factor can beimmobilized, and which is for performing B/F separation (separation of abound body and a free body), etc.

FIG. 6 shows an entire process in which a specimen is treated using atechnique of trapping and removing a nonspecific reaction factorutilizing an affinity gel to which an antibody against the nonspecificreaction factor is immobilized, and then a biologically-relevantsubstance is labeled using a technique of selectively collecting adesired biologically-relevant substance utilizing magnetic particles.Note that timing of the preparation of the second support is notrequired to be the same as that shown in FIG. 6. The preparation of thesecond support may be carried out during or before the step of removinga contaminant. FIG. 7 shows a case of carrying out different treatmentsaccording to an embodiment different from that shown in FIG. 6. FIG. 7shows an entire process in which a specimen is pretreated using atechnique of trapping and removing a nonspecific reaction factorutilizing magnetic particles to which an antibody against thenonspecific reaction factor is immobilized, and then abiologically-relevant substance is detected using a technique ofselectively collecting a desired biologically-relevant substanceutilizing magnetic particles. When the biologically-relevant substanceas an assay target is a nucleic acid, before a step of assaying a targetnucleic acid in a specimen, a step of extracting a nucleic acid using afirst support is carried out. Hereinafter, the respective steps such asthe pretreatment step and the assay step, etc. will be described in moredetail.

2. Biologically-Relevant Substance and Specimen

In the present invention, the “biologically-relevant substance” is asubstance which can be a detection target in the assay step, and meansevery biological substance such as a microorganism, a virus, a cell, anucleic acid, a polysaccharide, a simple protein, a complex protein anda low-molecular substance.

The microorganism includes a fungus, a eubacterium and anarchaebacterium. Examples of the fungus include microorganisms belongingto the genus Saccharomyces, the genus Aspergillus and the genus Candida.Examples of the eubacterium include microorganisms belonging to thegenus Mycobacterium, the genus Escherichia, the genus Bacillus, thegenus Listeria, the genus Vibrio, the genus Salmonella, the genusPseudomonas, the genus Staphylococcus, the genus Mycoplasma, the genusRickettsia and the genus Chlamydia. Examples of the archaebacteriuminclude microorganisms belonging to the genus Thermoplasma, the genusHalobacterium and the genus Methanobacterium. Specific examples thereofinclude Saccharomyces cerevisiae, Aspergillus nidulans, Candidaalbicans, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacteriumintracellulare, Mycobacterium kansasii, Escherichia coli, Bacilluscereus, Bacillus anthracis, Listeria monocytogenes, Vibrioparahaemolyticus, Vibrio cholerae, Salmonella typhi, Pseudomonasaeruginosa, Staphylococcus aureus, Mycoplasma pneumoniae, Rickettsiaprowazekii and Chlamydia trachomatis.

Examples of the virus include viruses belonging to Adenoviridae,Bacteriophage and Retroviridae. Specific examples thereof includeadenovirus, T7-like virus, hepatitis B virus, hepatitis C virus, humanimmunodeficiency virus, norovirus, human rota virus and influenza virus.Examples of the cell include an animal cell, a plant cell and an insectcell. Examples of the nucleic acid include a DNA, a RNA and anartificial nucleic acid. Examples of the polysaccharide include starch,glycogen, chitin and carrageenan. Examples of the protein include anantigen, an antibody, an enzyme, a chromoprotein and other polypeptides.Examples of the low-molecular substance include nucleotides such asnucleotide triphosphate and deoxynucleotide triphosphate, saccharidessuch as glucose and galactose, amino acids such as glutamic acid andlysine, dyes such as fluorescein and ethidium bromide, and hormones suchas epinephrine, peptide hormone and steroid. Note that theabove-described biologically-relevant substances are just examples andthe present invention is not limited to these substances. The specimenis not particularly limited as long as it contains such abiologically-relevant substance. The specimen includes, for example, (i)clinical materials such as sputum, expectorated sputum, saliva,mouthwash, stomach fluid, pleural lavage solution, blood, serum, plasma,feces, urine, spinal fluid and semen, (ii) biological materials such ascell lysate, tissue lysate, cell culture and tissue culture, (iii)effluents such as household effluent and industrial effluent, (iv)environmental water such as seawater, river water, pond water, lakewater and groundwater, and (v) drinking water, washing solution forfood, etc. and washing solution for tool in which abiologically-relevant substance may be present. The “washing solutionfor tool in which a biologically-relevant substance may be present”means a washing solution for a tool by which a portion to be confirmedwhether or not a biologically-relevant substance is present is wiped, ora washing solution by which a portion to be confirmed whether or not abiologically-relevant substance is present is washed. Examples thereofincludes a washing solution for a kitchen knife and a washing solutionfor a wiping cloth (a cloth for wiping the table) after used for wipinga thing.

In the present invention, various types of nonspecific reaction factorswhich are removed by the treatment of removing the contaminant areconsidered depending on the type of the specimen to be treated. Whenserum is used as the specimen, an immunoglobulin, a heterophilicantibody, a rheumatoid factor (RF) an M protein or the like may be thenonspecific reaction factor. The M protein is synonymous with amonoclonal immunoglobulin and means a protein observed when one type ofimmunoglobulin is increased. For example, an immunoglobulin proteinproduced by myeloma is an M protein which appears in serum of a patientsuffering from plasma cell dyscrasia typified by multiple myeloma.Depending on the type of myeloma producing M protein, M proteins areclassified into 5 types, i.e., IgA, IgM, IgG, IgE and Bence-Jonesprotein. The heterophilic antibody (HA) is a human anti-animal antibodyand is usually present in a healthy human at a rate of several percent.Examples of the heterophilic antibody include a human anti-mouseantibody (HAMA), a human anti-sheep antibody (HASA) and a humananti-goat antibody (HAGA). When such a heterophilic antibody is presentin a specimen, false-positive or false-negative nonspecific reaction isobserved in immunoassay using a mouse, goat, sheep or goat antibody.When a nucleic acid is to be detected from a specimen, the nucleic acidof interest can be detected using a complementary region of the nucleicacid or a nucleic acid comprising the complementary region instead of anantibody. For nucleic acid amplification used at the time of detection,for example, a PCR method can be applied, but there is no limitationthereon. Another suitable amplification method (an isothermalamplification method such as a LAMP method) may be used according topurpose.

3. Treatment of Specimen Using First Support

When using this treatment, the nonspecific reaction factor that causes afalse-positive reaction or false-negative reaction, which cannot beavoided by a conventional method in the field of immunoassay, can beremoved in advance. Therefore, the biologically-relevant substance canbe detected very specifically with high sensitivity. As the firstsupport, magnetic particles, a gel-like member, a membrane, a resinmember, corpus fibrosum or the like can be utilized, and they may besuitably selected according to need. As described hereinbelow, after thetreatment of removing the contaminant, the treated specimen is providedto an immunoassay step. Further, by this treatment, in the field ofnucleic acid assay, a target nucleic acid can be selectively extracted,and detection sensitivity at the time of assay can be improved. As thefirst support, it is possible to use magnetic particles or the like towhich a probe for trapping a nucleic acid is immobilized.

3-1. Treatment of Removing Contaminant Using Magnetic Particles to whichAntibody Against Nonspecific Reaction Factor is Immobilized

FIG. 8 shows a treatment of removing a contaminant using magneticparticles to which an antibody against a nonspecific reaction factor isimmobilized. As shown in FIG. 8, a pipette chip (dispensing chip) 10 isformed, for example, into an approximate elongated cylindrical shapewhich is tapered, and is detachably attached to the assay system. Themagnetic particles are used, for example, when separation, washing,suspension or the like is carried out in the pipette chip attached to adispensing nozzle. As shown in FIG. 8( a), the pipette chip 10 has a tipportion 10 a which is inserted into a well 12, a mounting portion 10 bwhich is fixed to a nozzle of the assay system (not shown) and a holdingportion 10 c which is formed between the tip portion 10 a and themounting portion 10 b and holds magnetic particles utilizing an externalmagnetic field. The inner diameter of the tip portion 10 a is smallest.The holding portion 10 c consists of, for example, a smaller diameterportion and a larger diameter portion. The inner diameter of the smallerdiameter portion is larger than the inner diameter of the tip portion,and the inner diameter of the larger diameter portion is smaller thanthe inner diameter of the mounting portion. Further, the inner diameterof the mounting portion 10 b is largest. It is desirable that the volumeof the pipette chip be suitably determined depending on the size of thewell. For example, when the volume is in the range of severalmicroliters to several hundred microliters, convenience may be improved.

The assay system for performing the treatment of removing thecontaminant has a magnet M, which is provided to the outer circumferenceof the holding portion 10 c to be allowed to move toward and away fromthe outer circumference and constrains magnetic particles by magneticforce, a nozzle to which the mounting portion 10 b of the pipette chip10 is attached, a pump mechanism for allowing the pipette chip 10attached to the nozzle to suck up or discharge a liquid, etc. In thisregard, the magnet M can constrain magnetic particles at the smallerdiameter portion of the holding portion 10 c (see FIGS. 6 and 7).

As shown in FIG. 8( b), when the pipette chip 10 is inserted into thewell 12, for example, a clearance of about 0.2 mm to 0.5 mm is providedbetween the pipette chip 10 and the well 12. By providing this, thecontact area between the specimen and the outside at the time ofinsertion of the pipette chip can be decreased as much as possible, andrisk of contamination is reduced. The shape of the pipette chip 10 maybe suitably changed, but the clearance between the well 12 and thepipette chip 10 at the time of insertion of the pipette chip 10 into thewell 12 is preferably narrower.

A magnetic particle 14 has magnetic property, and the size thereof is,for example, about 0.1 to 100 μm, and preferably about 0.1 to 10 μm. Thesize, mass, materials, structure, properties (paramagnetic property,superparamagnetic property, ferromagnetic property, ferrimagneticproperty, level of magnetic force), etc. of the magnetic particle 14 maybe arbitrarily determined according to the purpose of the treatment. Themagnetic particle can be formed using iron hydroxide, iron oxidehydrate, iron oxide, mixed iron oxide, iron, γ-Fe2O3, Fe3O4, etc.

FIG. 9 is a flow chart of a treatment of removing a contaminant usingmagnetic particles to which an antibody against a nonspecific reactionfactor is immobilized. As shown in FIG. 9, when the assay systemperforms a pretreatment of a specimen using the above-described well andpipette chip, firstly, a predetermined amount of the specimen held in awell is sucked up by the pipette chip 10. Next, the pipette chip 10 intowhich the specimen is sucked up is moved to the well 12 in which asolution of magnetic particles for pretreatment is held, and thespecimen in the pipette chip 10 is discharged into the well 12 in whichthe solution of magnetic particles for pretreatment is held. Using thepipette chip 10, the operation of mixing the specimen and the solutionof magnetic particles for pretreatment is repeated, and flow stirring iscarried out by sucking up and discharging, thereby producing ahomogeneous suspension. It is allowed to stand for required time afterstirring is completed, and a nonspecific reaction factor in the specimenis allowed to be bound to an antibody against the nonspecific reactionfactor immobilized to the magnetic body for pretreatment. After apredetermined time passes, a step of sucking up the suspension allowedto stand into the pipette chip 10 is carried out.

As shown in FIG. 8( c), the suspension sucked up into the pipette chip10 is held in the holding portion 10 c of the pipette chip 10. Themagnetic particle 14 for pretreatment suspended in the suspension isremotely fixed to a certain region on the inner wall surface of theholding portion 10 c by a magnetic field of the magnet M at the outsideof the pipette chip 10.

After the suspension is held in the pipette chip 10, the remainingsolution is discharged into the well 12 with the magnetic particle 14for pretreatment being fixed to one position by the magnetic field ofthe magnet M. In this way, the magnetic particle 14 for pretreatment towhich the nonspecific reaction factor 15 is bound is removed from thespecimen, thereby obtaining an assay sample in which the contaminant isremoved from the specimen. Thus, by utilizing the magnetic particle 14to which the antibody against the nonspecific reaction factor 15 isimmobilized for removal of the nonspecific reaction factor, thefrequency that the magnetic particle to which the antibody against thenonspecific reaction factor is immobilized encounters the nonspecificreaction factor can be increased, and it is possible to efficiently trapand remove the nonspecific reaction factor.

3-2. Treatment of Removing Contaminant Using Affinity Column to whichAntibody Against Nonspecific Reaction Factor is Immobilized

FIG. 10 shows a treatment of removing a contaminant using a column towhich an antibody against a nonspecific reaction factor is immobilized.FIG. 11 is a flow chart of a treatment of removing a contaminant using acolumn to which an antibody against a nonspecific reaction factor isimmobilized. As shown in FIGS. 10 and 11, a column-containing pipettechip 20 has a column 24 for removing a nonspecific reaction factor. Asshown in FIG. 10( a), the outer shape, size and material of thecolumn-containing pipette chip 20 are the same as those of theabove-described pipette chip 10. The column 24 contains many affinityresins 26 having a pellet-like shape, and to each affinity resin 26, anantibody for trapping the nonspecific reaction factor is bound. As shownin FIG. 10( b), when the specimen held in the well 12 is passed throughthe column 24, the nonspecific reaction factor in the specimen is boundto the above-described antibody and trapped by the column 24. A step ofsucking up the specimen into the column-containing pipette chip 20 andthen discharging the specimen from the column-containing pipette chip 20is repeated a predetermined number of times, thereby trapping morenonspecific reaction factors with the column 24. As shown in FIG. 10(c), by performing discharge into the well 12 after the step of suckingup and discharging the specimen is carried out a predetermined number oftimes, it is possible to provide an assay sample in which thenonspecific reaction factor has been removed from the specimen.

3-3. Treatment of Removing Contaminant Using Filtering Material Such asMembrane to which Antibody Against Nonspecific Reaction Factor isImmobilized

FIG. 12 shows a treatment of removing a contaminant using a membrane towhich an antibody against a nonspecific reaction factor is immobilized.FIG. 13 is a flow chart of a treatment of removing a contaminant using amembrane to which an antibody against a nonspecific reaction factor isimmobilized. As shown in FIGS. 12 and 13, a membrane-containing pipettechip 30 has a membrane 34 for removing a nonspecific reaction factor. Asshown in FIG. 12( a), the outer shape, size and material of themembrane-containing pipette chip 30 are the same as those of theabove-described pipette chip 10. The membrane 34 is formed into asheet-like shape, and an antibody against the nonspecific reactionfactor is immobilized to the membrane 34. As shown in FIG. 12( b), whenthe specimen is passed through the membrane 34, the nonspecific reactionfactor in the specimen is bound to the above-described antibody andtrapped by the membrane 34. A step of sucking up the specimen in thewell 12 into the membrane-containing pipette chip 30 and thendischarging the specimen from the membrane-containing pipette chip 30 isrepeated a predetermined number of times, thereby trapping morenonspecific reaction factors with the membrane 34. As shown in FIG. 12(c), by performing discharge into the well 12 after the step of suckingup and discharging the specimen is carried out a predetermined number oftimes, it is possible to provide an assay sample in which thenonspecific reaction factor has been removed from the specimen.

3-4. Treatment of Removing Contaminant Using Gel to which Reducing Agentis Immobilized

FIG. 14 shows a treatment of removing a contaminant using a gel to whicha reducing agent is immobilized. FIG. 15 is a flow chart of a treatmentof removing a contaminant using a gel to which a reducing agent isimmobilized. As shown in FIGS. 14 and 15, a gel-containing pipette chip40 has a gel 43 for decomposing a nonspecific reaction factor. As shownin FIG. 14( a), the outer shape, size and material of the gel-containingpipette chip 40 are the same as those of the above-described pipettechip 10. To this gel 43, a reducing agent for decomposing a nonspecificreaction factor (substance that inactivates a contaminant) isimmobilized. As the reducing agent, for example,Tris(2-carboxylethyl)phosphine (TCEP), glutathione or the like can beused. As shown in FIG. 14( b), when the specimen is passed through thegel to which the reducing agent is immobilized, the nonspecific reactionfactor in the specimen is decomposed by the reducing agent. A step ofsucking up the specimen in the well 12 into the gel-containing pipettechip 40 and then discharging the specimen from the gel-containingpipette chip 40 is repeated a predetermined number of times, therebydecomposing more nonspecific reaction factors with the reducing agent.As shown in FIG. 14( c), by performing discharge into the well 12 afterthe step of sucking up and discharging the specimen is carried out apredetermined number of times, it is possible to provide the specimenfrom which the nonspecific reaction factor has been removed. Theexplanation above is about an example in which the nonspecific reactionfactor is decomposed by the reducing agent held by the gel, but anantibody against the nonspecific reaction factor may be held by the gel.In this case, using the gel to which the antibody against thenonspecific reaction factor is immobilized, the nonspecific reactionfactor can be trapped and removed.

3-5. Other Cases (Treatment Using Plastic Member)

In addition to the above-described embodiments, for example, it is alsopossible to pretreat the specimen using a product in which the antibodyagainst the nonspecific reaction factor is immobilized to a plasticsupport. For example, a plurality of concave holes are arranged on asupport in a matrix fashion, and the antibody against the nonspecificreaction factor is immobilized in the concave holes in advance. When thenonspecific reaction factor in the specimen is held in the concaveholes, the nonspecific reaction factor in the specimen is bound to theantibody against it and immobilized. By immersing the tip portion of thepipette chip in the concave holes to suck up a liquid, it is possible toprepare an assay sample in which the nonspecific reaction factor hasbeen removed from the specimen.

3-6. Treatment of Extracting Target Nucleic Acid Using MagneticParticles to which Probe for Trapping Nucleic Acid is Immobilized

FIG. 16 is an explanatory drawing for schematically explaining atreatment of extracting a target nucleic acid using magnetic particles(first support) to which a probe for trapping the nucleic acid isimmobilized. FIG. 17 is a flow chart of a treatment of extracting atarget nucleic acid using magnetic particles to which a probe fortrapping the nucleic acid is immobilized. As shown in FIG. 16, when theassay system pretreats a specimen using a well and a pipette chip,firstly, a predetermined amount of the specimen held in a well is suckedup into a pipette chip 220. Next, the pipette chip 220 into which thespecimen is sucked up is moved to a well 222 in which a solution ofmagnetic particles for pretreatment is held, and the specimen in thepipette chip 220 is discharged into the well 222 in which the solutionof magnetic particles for pretreatment is held. Using the pipette chip220, the specimen and the solution of magnetic particles forpretreatment are mixed homogeneously by sucking up and discharging,thereby producing a suspension. It is allowed to stand for apredetermined time after stirring is completed, and a target nucleicacid 225 in the specimen is allowed to be bound to a probe to which amagnetic particle 224 for pretreatment is immobilized.

As shown in FIG. 16( c), after the suspension is allowed to stand for apredetermined time, the suspension is sucked up into the pipette chip220 and held in a holding portion 220 c. The magnetic particle 224 forpretreatment contained in the suspension is remotely fixed to a certainregion on the inner wall surface of the holding portion 220 c by amagnetic field of a magnet M at the outside of the pipette chip 220.After the suspension is held in the pipette chip 220, the remainingsolution is discharged into the well 222 with the magnetic particle 224for pretreatment being fixed to one position by the magnetic field ofthe magnet M. The magnetic particle 224 to which a target nucleic acid225 is bound is taken out from the specimen, thereby extracting thetarget nucleic acid from the specimen. Thus, by using magnetic particlesto which a probe which can bind to a target nucleic acid is immobilizedfor extraction of the target nucleic acid, the target nucleic acid canbe trapped and extracted efficiently.

On a well plate, for example, a plurality of wells 12 are arranged in aline or in a matrix fashion. The specimen is held in a specific well 12in advance, and in another well, a solution containing a required amountof magnetic particles (hereinafter referred to as “magnetic particlesfor pretreatment”) to which an antibody against a nonspecific reactionfactor (hereinafter referred to as “antibody for pretreatment”) isimmobilized (hereinafter referred to as “solution of magnetic particlesfor pretreatment” is held in advance.

In the present invention, before moving to a step of detecting an assaysample, a step of preparing a second support to which a substance havingaffinity to a biologically-relevant substance is immobilized is carriedout. FIG. 18 is an explanatory drawing for schematically explaining amagnetic particle to which an antibody against an antigen isimmobilized, a plate to which the antibody against the antigen isimmobilized, and a bead to which the antibody against the antigen isimmobilized. As the second support to be prepared before moving to adetection step, for example, as shown in FIG. 18, (a) a magneticparticle G to which an antibody 46 against an antigen 45 is immobilized,(b) a plate P to which the antibody 46 against the antigen 45 isimmobilized, and (c) a bead B to which the antibody 46 against theantigen 45 is immobilized are exemplified. As shown in FIG. 18( a), asecond support can be prepared by immobilizing the antibody 46 againstthe antigen 45 to the magnetic particle G. Further, as shown in FIG. 18(b), a second support of another embodiment can be prepared byimmobilizing the antibody 46 against the antigen 45 to the plate P.Moreover, as shown in FIG. 18( c), a second support of yet anotherembodiment can be prepared by immobilizing the antibody 46 against theantigen 45 to the bead B. By preparing the second support in advance inthis way, it is possible to smoothly carry out a next assay step, andimprovement of assay accuracy, etc. can be expected. To the preparedsecond antibody, the antigen 45 can be specifically bound, and inaddition, to the antigen 45, a labeling substance 47 that causes afluorescence reaction or luminescence reaction can be specifically boundvia another antibody against the antigen (secondary antibody).

4. Assay of Biologically-Relevant Substance

The step of assaying a biologically-relevant substance includes a stepof labeling the biologically-relevant substance and a step of detectingthe labeled biologically-relevant substance. Hereinafter, each step willbe described.

4-1. Labeling Reaction Step

A labeling reaction step is included in the step of assaying a targetbiologically-relevant substance and is carried out using the secondantibody. FIG. 19 is an explanatory drawing for schematically explainingan embodiment in which an antigen is detected by trapping and labelingthe antigen using a second support. As shown in FIG. 19, in the labelingreaction step, a biologically-relevant substance is trapped from anassay sample in which treatments such as removal of a contaminant from aspecimen have been performed and the biologically-relevant substance islabeled. For example, when an antigen is to be trapped as thebiologically-relevant substance, for labeling the antigen, the antibody46 that binds to the antigen 45 is used. After the labeling substance 47is bound to the antigen 45, a detection step described hereinbelow iscarried out. As shown in FIGS. 19 (a) and (b), in the case where themagnetic particle G to which the antibody 46 against the antigen 45 isimmobilized is used as the second support, and in the case where theplate P to which the antibody 46 against the antigen 45 is immobilizedis used as the second support, for example, the labeled antigen can bedetected using a photomultiplier tube (PMT) 48. Further, as shown inFIG. 19 (c), in the case where the bead B to which the antibody 46against the antigen 45 is immobilized is used as the second support, forexample, the antigen can be detected using a photon counter utilizingoptical fiber. Hereinafter, two typical embodiments in which an antigenis trapped from a specimen and a labeling antibody is provided theretowill be described more specifically.

4-1-1. Labeling of Antigen Using Magnetic Particles to which AntibodyAgainst Antigen is Immobilized

Here, an embodiment in which a single antigen is trapped and labeledusing magnetic particles to which an antibody against the antigen isimmobilized as the second support will be described. After the treatmentof highly purifying the specimen, the assay system carries out animmunoassay having the assay step comprising the labeling reaction step.In a first well (first holding portion) on a well plate, a solutioncontaining a required amount of magnetic particles (hereinafter referredto as “magnetic particles for specific reaction”) to which an antibodyagainst an antigen targeted for detection (hereinafter referred to as“antibody for specific reaction”) is immobilized (hereinafter referredto as “solution of magnetic particles for specific reaction”) is held inadvance, and in addition, in a second well (second holding portion), asolution containing a labeling antibody against the antigen (hereinafterreferred to as “labeling antibody”) (hereinafter referred to as“labeling solution”) is held in advance. In addition, in a third well, asubstrate solution is held.

FIG. 20 shows an immunoassay using magnetic particles. As shown in FIG.20, another pipette chip 50, which is the same type of the pipette chipused in the pretreatment such as removal of the contaminant, is newlyattached to the nozzle, and the solution of magnetic particles forspecific reaction held in another well is sucked up into the pipettechip 50. As shown in FIG. 20( a), the pipette chip 50 holding thesolution of magnetic particles for specific reaction inside iscontrolled to be moved to a well 12 in which an assay sample is held,and the tip portion is immersed in the well 12. The magnet M isgradually moved away from the pipette chip 50 to release the magneticparticles for specific reaction from constraining by the magnetic field,and the solution of magnetic particles for specific reaction is mixedwith the assay sample. A mixture of the solution of magnetic particlesfor specific reaction and the assay sample is sucked up and discharged,thereby forming a suspension in which the magnetic particles forspecific reaction are homogeneously suspended. As shown in FIG. 20( a)and FIG. 20( b), after the suspension is formed, for example, it isallowed to stand (incubated) at 37° C. for a certain period of time, andthe antigen in the suspension is specifically reacted with and bound tothe antibody for specific reaction immobilized to the magnetic particlesfor specific reaction. In the explanation above, the solution ofmagnetic particles for specific reaction is sucked up into the pipettechip 50 and added to the specimen holding portion (well) 12 in which theassay sample is held, but it is also possible to suck up the assaysample into the pipette chip 50 and add the assay sample to a well inwhich the solution of magnetic particles for specific reaction is held.

As shown in FIG. 20( b), after the suspension is allowed to stand, thesuspension is held in the pipette chip 50. After the suspension is heldin the pipette chip 50, the magnet M is moved toward the outercircumference of the holding portion of the pipette chip 50, and themagnetic particles for specific reaction to which the antigen is bound(hereinafter referred to as “antigen-bound magnetic particles”) aregathered at one position in the holding portion of the pipette chip 50.After the antigen-bound magnetic particles are collected, the remainingsolution is discharged into the well 12, and only the antigen-boundmagnetic particles are held in the pipette chip 50.

As shown in FIG. 20( b), the pipette chip 50 holding the antigen-boundmagnetic particles is controlled to be moved to a well 60 holding awashing solution with the antigen-bound magnetic particles beingmaintained to be held. The tip portion of the pipette chip 50 isimmersed in the washing solution in the well 60, and after that, themagnet M is gradually moved away from the outer circumference of theholding portion of the pipette chip 50, and the antigen-bound magneticparticles in the pipette chip 50 are mixed with the washing solution.The washing solution with which the antigen-bound magnetic particles aremixed is subjected to flow stirring by sucking up and discharging by thepipette chip 50. After stirring, the washing solution with which theantigen-bound magnetic particles are mixed is sucked up into the pipettechip 50. The magnet M is moved toward the outer circumference of theholding portion of the pipette chip 50, and the antigen-bound magneticparticles are gathered at one position. After the antigen-bound magneticparticles are constrained at one position, the remaining solution isdischarged into the well 60.

As shown in FIG. 20( c), after the solution is discharged, the pipettechip 50 with the antigen-bound magnetic particles being held iscontrolled to be moved to a well 62 holding a labeling solutioncontaining a labeling antibody (enzyme-labeling antibody) against anantigen. The tip portion of the pipette chip 50 is immersed in thelabeling solution, and then the magnet M is gradually moved away fromthe outer circumference of the holding portion of pipette chip 50 torelease the antigen-bound magnetic particles from constraining. Bysucking up and discharging the labeling solution with which theantigen-bound magnetic particles are mixed, the antigen-bound magneticparticles can be mixed with and homogeneously suspended in the labelingsolution. After suspending, for example, the obtained suspension isallowed to stand at 37° C. for a certain period of time, therebyallowing the enzyme-labeling antibody to bind to the antigen.

As shown in FIG. 20( d), after the suspension is allowed to stand(incubated) for a certain period of time, the suspension in the well 62is slowly sucked up into the pipette chip 50. After the suspension isheld in the pipette chip 50, the magnet M is moved toward the pipettechip 50, and the magnetic particles suspended in the suspension held areconstrained at one position. After constraining the magnetic particlesto which the enzyme-labeling antibody is bound (hereinafter referred toas “labeling antibody-bound magnetic particles”), the solution fromwhich the labeling antibody-bound magnetic particles are removed isdischarged into the well 62, and only the labeling antibody-boundmagnetic particles remain in the pipette chip 50.

After that, as shown in FIG. 20( e), the pipette chip 50 with thelabeling antibody-bound magnetic particles being held is controlled tobe moved to a well 64 holding a washing solution. The magnet M isgradually moved away from the pipette chip 50, and the washing solutionin the well 64 is mixed with the labeling antibody-bound magneticparticles (see FIG. 20( f)). The labeling antibody-bound magneticparticles are washed in a manner similar to that of thealready-described washing step, and after that, the pipette chip 50holding the labeling antibody-bound magnetic particles is controlled tobe moved to a well 67 holding a substrate solution, and a detection stepdescribed hereinbelow (see FIG. 20( g)) is started.

In the explanation above, the magnetic particles to which the antibodyagainst the antigen is immobilized are mixed with the specimen after thetreatment such as removal of the contaminant to allow the antibody tobind to the antigen, and after that, using the labeling solution, theenzyme-labeling antibody is bound to the antigen. However, the order oflabeling is not limited thereto. For example, it is possible to usemagnetic particles to which the enzyme-labeling antibody and theantibody are immobilized in advance.

4-1-2. Simultaneous Labeling Reaction Step of a Plurality of Types ofAntigens Using a Plurality of Antibody-Immobilized Beads

In item [4-1-1] above, a single antigen is trapped and labeled, but inthis item, an embodiment in which a plurality of types of antigens aretrapped and labeled at a time will be described. FIG. 21 shows animmunoassay using an antigen separation/immobilization tube. As shown inFIG. 21, a transparent antigen separation/immobilization tube 70 formedinto a tubular shape holds beads as the second support to which anantibody against an antigen is immobilized in advance (hereinafterreferred to as “antibody-immobilized beads”) and spacer beads 72 whichare positioned so that a certain number of antibody-immobilized beadsare separated, and these beads are arranged in a line along the tube.Regarding the antibody-immobilized beads, for example, 3 firstantibody-immobilized beads 74 to which a first antibody is immobilized,3 second antibody-immobilized beads 76 to which a second antibody isimmobilized, and 3 third antibody-immobilized beads 78 to which a thirdantibody is immobilized are respectively positioned continuously, andbetween the continuously-positioned antibody-immobilized beads 74, 76and 78, each spacer bead 72 is positioned. The arrangement of the beadsmay be suitably changed, and in some cases, the spacer beads can beomitted.

To the upper end portion of the antigen separation/immobilization tube70, a mounting portion (not shown) to be mounted on the nozzle of theassay system is provided, and the lower end is opened so that a liquidcan be sucked up and discharged. A pump mechanism is provided to theassay system of the present invention so that a liquid can be sucked upinto or discharged from the antigen separation/immobilization tube 70mounted on the nozzle.

As shown in FIG. 21( a), when the lower end of the antigenseparation/immobilization tube 70 is immersed in a well 12 and an assaysample in the well 12, which has been subjected to treatments such asremoval of a contaminant, is sucked up into the antigenseparation/immobilization tube 70, first to third antigens, whichrespectively correspond to the first to third antibodies, bind to theantibodies and are trapped by the beads 74, 76 and 78 to which the firstto third antibodies are immobilized respectively. By repeating suckingup and discharging of the specimen a predetermined number of times, theantigens are certainly bound to the antibody-immobilized beads 74, 76and 78.

As shown in FIG. 21( b), after repeating sucking up and discharging ofthe specimen a predetermined number of times, a washing solution inanother well 80 is sucked up and the first to thirdantibodies-immobilized beads 74, 76 and 78 are washed. As shown in FIG.21( c), after washing the first to third antibodies-immobilized beads74, 76 and 78, the lower end of the antigen separation/immobilizationtube 70 is immersed in an enzyme-labeling solution held in another well84, and the enzyme-labeling solution is sucked up into the antigenseparation/immobilization tube 70. In the enzyme-labeling solution, 3types of enzyme-labeling antibodies, which correspond to the respectiveantigens that bind to the first to third antibodies, are mixed. When theenzyme-labeling solution is sucked up into the antigenseparation/immobilization tube 70, the enzyme-labeling antibodies bindto the respective antigens. As shown in FIG. 21( d), after carrying outsucking up and discharging of the enzyme-labeling solution apredetermined number of times, a washing solution held in another well84 is sucked up and discharged, thereby washing the first to thirdantibodies-immobilized beads 74, 76 and 78 to which the antigens and theenzyme-labeling antibodies are bound. As shown in FIG. 21( e), afterwashing, the antigen separation/immobilization tube is controlled to bemoved to a well 86 holding a substrate solution, and a detection stepdescribed hereinbelow is started. In the explanation above, the mixtureof the 3 types of the enzyme-labeling antibodies is used, butalternatively, enzyme labeling can be performed by using 3 wells, inwhich different types of enzyme-labeling antibodies are heldrespectively, and performing a step consisting of sucking up anddischarging of an enzyme-labeling antibody and washing with respect tothese 3 wells sequentially.

Thus, by treating the specimen using the antigenseparation/immobilization tube 70 in which the beads 74, 76 and 78 towhich the different types of antibodies are respectively immobilized arearranged, a plurality of antigens can be trapped at a time, and manyitems can be detected simultaneously. In addition, it is possible toreduce the time to detect the antigen in the specimen.

In the explanation above, the beads 74, 76 and 78 to which theantibodies against the antigens are immobilized are contacted with thespecimen that has been subjected to treatments such as removal of acontaminant to allow the antibodies to bind to the antigens, and thenthe enzyme-labeling antibodies are bound to the antigens using thelabeling solution. However, the order of labeling is not limitedthereto. For example, it is possible to perform a step of trappingantigens using beads to which enzyme-labeling antibodies are immobilizedin advance.

4-2. Detection Step

The antigens to which the enzyme-labeling antibodies are bound are mixedwith the substrate solution to develop the color of the substrate, anddetection of absorbance, etc. is carried out. Hereinafter, a detectionstep in the case where antigens are labeled using the above-describedmagnetic particles and a detection step in the case where antigens arelabeled using the antigen separation/immobilization tube will bedescribed separately.

4-2-1. Detection Utilizing Magnetic Particles and Pipette Chip

The assay system of the present invention has, for example, a lightirradiation portion which irradiates the side of a well with a lightflux having a specific wavelength, a light receiving portion whichreceives the light flux irradiated from the light irradiation portionvia the well, a signal processing circuit which processes a signaloutputted from the light receiving portion to form, for example,absorbance data or emission intensity data, etc.

As shown in FIG. 20( g), the pipette chip 50 holding the labelingantibody-bound magnetic particles is controlled to be moved to the well67 holding the substrate solution. After the tip portion of the pipettechip 50 is immersed in the substrate solution in the well 67, the magnetM is moved away from the outer circumference of the holding portion ofthe pipette chip 50 to release the labeling antibody-bound magneticparticles from constraining, and the labeling antibody-bound magneticparticles are mixed with the substrate solution. After the labelingantibody-bound magnetic particles are mixed with the substrate solution,sucking up the mixture into the pipette chip 50 and discharging themixture into the well 67 are carried out a predetermined number oftimes, thereby forming a suspension in which the labeling antibody-boundmagnetic particles are dispersed homogeneously. In this way, thelabeling antibody-bound magnetic particles can be homogeneously reactedwith the substrate solution.

The labeling antibody-bound magnetic particles are reacted with thesubstrate solution to develop the color of the substrate, and afterthat, for example, the side of the well 67 is irradiated with a lightflux having a specific wavelength, and absorbance thereof is detected.Note that in the case of a test method in which a luminescent state ismaintained for a very short time, such as CLIA, the following method maybe employed: a liquid-holding portion is provided; a filter and awater-absorbing pad are provided to the liquid-holding portion; themagnetic particles are discharged together with the washing solutionsucked up in the previous step from the pipette chip into theliquid-holding portion and the magnetic particles are collected by thefilter; after that, a luminescence-inducing solution such as hydrogenperoxide solution (H₂O₂) is supplied from the nozzle to allow themagnetic particles to become luminescent; and luminescence at the timeof dispensing is measured using an optical measuring device such as PMT.

4-2-2. Detection Utilizing Antigen Separation/Immobilization Tube

As shown in FIG. 21( e), when a plurality of antigens are trapped usingthe antigen separation/immobilization tube 70 as the second support, theplurality of the antigens can be detected at a time. The assay system ofthe present invention has a plurality of light irradiation portionswhich irradiate with a light flux having a specific wavelength, aplurality of light receiving portions which receive the light fluxirradiated from each of the light irradiation portions via the antigenseparation/immobilization tube 70, a signal processing circuit whichforms emission intensity data by, for example, amplifying and digitizingoutput signals from the light receiving portions, etc. Firstly, thelower end of the antigen separation/immobilization tube 70 is immersedin the well 86 holding the substrate solution, and the substratesolution is sucked up into the antigen separation/immobilization tube70. Sucking up and discharging the substrate solution is carried out apredetermined number of times to sufficiently perform a luminescentreaction. The light irradiation portions and the light receivingportions are, for example, provided so as to correspond to theantibody-immobilized beads 74, 76 and 78. The light irradiation portionsand the light receiving portions are arranged to be opposed to eachother via the antibody-immobilized beads 74, 76 and 78. Light fluxesfrom the light irradiation portions are respectively received by thelight receiving portions via the beads 74, 76 and 78 in the antigenseparation/immobilization tube 70. Based on the output signals from thelight receiving portions, for example, absorbance data or emissionintensity data regarding each of the beads 74, 76 and 78 is formed.

Assay System

5-1. Immunoassay System

The present invention provides an assay system comprising a pretreatmentmeans for pretreating a specimen and an immunoassay means for performingan immunoassay of the specimen pretreated by the pretreatment means, andthe immunoassay means comprises a labeling reaction means and adetection means. The assay system of the present invention is totallyautomated from the step of pretreating a specimen to the detection step,and a biologically-relevant substance in the specimen can be detectedautomatically. As already described above, regarding each of thepretreatment step and the assay step (a labeling reaction may beincluded), there are a plurality of variations, and the assay system isconstituted according to the combination of such variations of thepretreatment step and the assay step.

As described above, as the means for removing a contaminant, which isthe feature of the present application, the following 5 main embodimentscan be employed: (i) a means for trapping and removing a nonspecificreaction factor utilizing magnetic particles to which an antibodyagainst the nonspecific reaction factor is immobilized; (ii) a means fortrapping and removing a nonspecific reaction factor utilizing anaffinity gel to which an antibody against the nonspecific reactionfactor is immobilized; (iii) a means for trapping and removing anonspecific reaction factor utilizing a filter to which an antibodyagainst the nonspecific reaction factor is immobilized; (iv) a means fortrapping and removing a nonspecific reaction factor utilizing a plasticto which an antibody against the nonspecific reaction factor isimmobilized; and (v) a means for decomposing a nonspecific reactionfactor using a gel to which a reducing agent is immobilized. As thelabeling reaction step, the following 2 main embodiments can beemployed: (i) a labeling reaction means utilizing magnetic particles anda pipette chip; and (ii) a labeling reaction means utilizing an antigenseparation/immobilization tube.

The detection step is selected depending on the means selected for thelabeling reaction means. For example, when (i) a labeling reaction meansutilizing magnetic particles and a pipette chip is selected for thelabeling reaction means, a detection means utilizing magnetic particlesand a pipette chip is preferably selected, and when (ii) a labelingreaction means utilizing an antigen separation/immobilization tube isselected for the labeling reaction means, a detection means utilizing anantigen separation/immobilization tube is preferably selected. Further,it is also possible to produce an assay system which has both (i) alabeling reaction means utilizing magnetic particles and a pipette chipand (ii) a labeling reaction means utilizing an antigenseparation/immobilization tube. However, in this case, a mechanism ofchange between the pipette chip and the antigenseparation/immobilization tube to be attached to a nozzle is furtherprovided to the assay system, or both a nozzle for the pipette chip anda nozzle for the antigen separation/immobilization tube are provided tothe assay system. Thus, it is possible to design at least 10 types ofassay systems depending on the combination of the types of the means forremoving the contaminant, the labeling reaction means and the detectionmeans. The assay system may be produced with suitable modificationaccording to the purpose of use, etc. Hereinafter, a particularlypreferred embodiment will be described.

FIG. 22 is a block diagram of an assay system in which the pretreatmentstep is carried out and then the assay step is carried out. Hereinafter,the system utilizing magnetic particles will be described. An assaysystem 100 has a central processing unit 102, a chip position controller104, a chip mount controller 106, a magnetic field controller 108, atemperature controller 110, a pumping controller 112, a timer 114, a RAM116, a ROM 118, a display panel 120, an operation interface 122, etc.

The chip position controller 104 has mutually orthogonal axes X, Y andZ, and the position of the nozzle is controlled by a stepping motor or aservomotor. The axes X and Y are approximately parallel to a well plateand mutually orthogonal, and the axis Z is approximately perpendicularto the well plate. At the time of the movement of the nozzle, forexample, the nozzle is moved in two steps, i.e., a movement on the axesX and Y that are approximately parallel to the well plate, and amovement on the axis Z that is approximately perpendicular to the wellplate.

In the ROM 118, various control programs are stored. According to anoperation mode selected by a user via the operation interface 122, acontrol program is developed from the ROM 118 to RAM 116, and thecentral processing unit 102 controls each portion of the system 100based on the control program developed in the RAM 116.

The display panel 120 displays items required to be provided to theuser. For example, the display panel 120 can display the number of timesof pumping at the time of the treatment of removing the contaminant inthe specimen, time to be allowed to stand after suspension of themagnetic particles, the flow rate at the time of pumping, the amount tobe sucked up and discharged, the rate of movement of the pipette chip,etc., and the user can confirm these items by the display. When setcontents are desired to be changed, they can be changed by operation ofthe operation interface 122.

The timer 114 carries out timing according to a program read from theROM 118. Timing is carried out, for example, when incubation or pumpingis performed. By timing, each step is carried out accurately.

The magnetic field controller 108 manages the placement of the magnet130 to control the strength of the magnetic field provided to thepipette chip. The magnetic field controller 108 has mutually orthogonalaxes X, Y and Z, and the placement of the magnet 130 is managed by astepping motor or a servomotor. The axes X and Y are approximatelyparallel to a well plate and mutually orthogonal, and the axis Z isapproximately perpendicular to the well plate. At the time of themovement of the magnet 130, for example, the placement of the magnet 130can be adjusted in two steps, i.e., a movement on the axes X and Y thatare approximately parallel to the well plate, and a movement on the axisZ that is approximately perpendicular to the well plate. Usually, onlythe movement on the axes X and Y is carried out, but optionally, themovement on the axis Z can be carried out.

The temperature controller 110 has a heater 136, a thermal sensor 138,etc., and manages the temperature of the liquid held in the pipettechip. The heater 136 is allowed to produce heat by electric powersupplied by the temperature controller 110. The thermal sensor 138transmits a temperature signal to the temperature controller 110depending on the temperature of the liquid held in the pipette chip. Thetemperature controller 110 detects the temperature based on thetemperature signal from the thermal sensor 138 and adjusts the electricpower supplied to the heater 136.

The chip mount controller 106 performs attachment of the pipette chip tothe nozzle and detachment of the pipette chip from the nozzle. The chipmount controller 106 is placed at a position which is remote from thewell plate to some extent, so that contamination is prevented if theliquid is spattered from the pipette chip at the time of exchange of thepipette chip. The chip mount controller 106 has a gripping portion forgripping the pipette chip and a chip preparation portion for preparinganother new pipette chip. When the nozzle is moved upward along the axisZ with the pipette chip being gripped by the gripping portion, thepipette chip is detached from the nozzle. Next, the bared nozzle ismoved on the axes X and Y to move to a position above a new pipettechip. At the chip preparation portion, the new pipette chip is held witha mount portion side up and a tip portion side down. When the nozzle ismoved downward along the axis Z, the mount portion of the new pipettechip is attached to the nozzle. Examples of embodiments of engagementbetween the nozzle and the pipette chip include an engagement formutilizing a latch and a notch with which the latch is engaged, anengagement form utilizing a boss and a rib, and an engagement formutilizing a male screw and a female screw. Any suitable engagement formmay be selected.

The pumping controller 112 has a pump 140 and a pressure sensor 146, andcontrols sucking up and discharging of the liquid performed via thenozzle and the pipette chip attached to the nozzle. The pump 140 has ahousing formed into a cylindrical shape, a piston that is movably fittedinto the housing and a motor for driving the piston. The inside of thehousing communicates with the opening of the nozzle. The movement of thepiston is controlled, for example, by a servomotor, and driving of theservomotor is controlled by a drive control signal from the pumpingcontroller 112. When the piston is activated, it becomes possible tosuck up or discharge the liquid through the opening of the nozzle.

In the opening of the nozzle, a pressure sensor 146 for detecting thepressure is provided, and the pressure sensor 146 transmits a pressuresignal to the pumping controller 112. The pumping controller 112monitors the pressure based on the pressure signal from the pressuresensor 146. In this constitution, for example, when the tip portion ofthe pipette chip is immersed in the specimen in the well, the pressuredetected by the pumping controller 112 exceeds a predeterminedthreshold, and in response to this, the drive control signal istransmitted to the servomotor. Also at the time of sucking up anddischarging the specimen, the pressure sensor 146 constantly transmitsthe pressure signal to the pumping controller 112. Therefore, thepumping controller 112 can control driving of the servomotor with highaccuracy, and monitors levels of the pressure of sucking up the specimenand the pressure of discharging the specimen, thereby performingmanagement so as to allow sucking up and discharging to be carried outwithin a predetermined range. Note that the means for the treatment ofremoving the contaminant is constituted by the magnetic field controller108, the pumping controller 112, the magnetic particles to which theantibody against the nonspecific reaction factor is immobilized, etc.The means for stirring is constituted by the pipette chip, the pumpingcontroller 112, the pump 140, the pressure sensor 146, etc. Further, themeans for separation is constituted by the magnetic field controller108, the magnet 130, etc.

The action of the above-described constitutions will be described. Whenthe step of the treatment of removing the contaminant is started, thetip portion of the pipette chip is immersed in the specimen in the well12, and based on the pressure signal from the pressure sensor 146, thepump 140 is activated. After the specimen is sucked up into the pipettechip, the pipette chip is controlled to be moved to the well 12 in whichthe solution of magnetic particles for pretreatment is held, and the tipportion of the pipette chip is immersed in the solution of magneticparticles for pretreatment. Based on the pressure signal from thepressure sensor 146, immersion of the tip portion of the pipette chip isdetected, and then the piston of the pump 140 is activated to startpumping. When the pumping is started, the magnetic particles to whichthe antibody against the nonspecific reaction factor is immobilized aredispersed, thereby forming a suspension.

After a predetermined amount of time passes after the formation of thesuspension, the pump 140 is activated and the suspension is sucked upinto the pipette chip 10. After the suspension is sucked up into thepipette chip 10 and the pump 140 is stopped, the magnet 130 is movedtoward the holding portion of the pipette chip and the magneticparticles for pretreatment are fixed to one position on the inner wallsurface. The pump 140 is activated with the magnetic particles forpretreatment being fixed to one position by the magnet, therebydischarging the liquid into the well. In this way, the contaminant isremoved and the magnetic particles for pretreatment to which thenonspecific reaction factor is bound are separated from the specimen toprepare an assay sample to be assayed.

After obtaining the assay sample in which the contaminant contained inthe specimen has been removed, magnetic particles for specific reactionare added to the assay sample to extract the antigen from the specimen.Further, antigen-bound magnetic particles are labeled with theenzyme-labeling antibody to obtain labeling antibody-bound magneticparticles. The obtained labeling antibody-bound magnetic particles areadded to the substrate solution to detect absorbance, etc.

Thus, by pumping the specimen mixed with the magnetic particles to whichthe antibody against the nonspecific reaction factor is immobilized, thespecimen is stirred and the magnetic particles move in the specimen.Therefore, the frequency that the magnetic particles to which theantibody against the nonspecific reaction factor is immobilizedencounter the nonspecific reaction factor in the specimen is increased,and the nonspecific reaction factor can be bound to the antigen againstthe nonspecific reaction factor more certainly. Further, by separatingthe magnetic particles to which the nonspecific reaction factor is boundfrom the specimen using the magnet, the nonspecific reaction factor canbe efficiently removed from the specimen. In addition, the steps fromthe pretreatment of the specimen to the immunoassay can be carried outcollectively and continuously, and a highly convenient system can beprovided to users. Moreover, since the pretreatment of the specimen iscarried out in accordance with the pumping mechanism, chip and well usedin the immunoassay step, the pumping mechanism and the like can be usedfor two purposes when constructing a system in which the pretreatmentstep is integrated into the immunoassay step, and therefore, it ispossible to prevent the system from becoming too enormous.

Further, as described above, the movement of the piston driven at thetime of pumping is controlled by the servomotor based on the pressuresignal from the pressure sensor 146. Therefore, the amount of the liquidsucked up at the time of pumping, the amount of the liquid discharged,the pressure of sucking up and the pressure of discharging can becontrolled with high accuracy, and the flow of the liquid can be rapidlycontrolled. Therefore, dispersion of the magnetic particles as describedabove can be carried out in a short time, and reduction in pretreatmenttime and the like can be realized. Further, since pumping of thespecimen is carried out in a state in which the opening at the tipportion of the pipette chip is immersed in the specimen, bubbling of thespecimen can be reduced, and inclusion of atmosphere in contact with thespecimen in the specimen can be reduced.

Further, pumping is carried out using a pipette chip and a well, and thesizes of the pipette chip and well may correspond to the sizes of thepipette chip and well used in the labeling reaction step or the assaystep. By using the pipette chips of the same size and the wells of thesame size, size reduction in the assay system can be expected. In theexplanation above, the magnetic particles to which the antibody againstthe nonspecific reaction factor is immobilized are used, but amicro-sized small sphere to which the nonspecific reaction factor isimmobilized may also be used. In this case, by using a filter or thelike having a mesh size with which the small sphere cannot be passedthrough, the small sphere to which the nonspecific reaction factor isimmobilized can be separated from the specimen.

The assay system in which the labeling reaction step is carried outusing the magnetic particles to which the antibody against the antigenis immobilized basically has the same constitution as that shown in FIG.22, except that the pattern of movement of the magnet and the like aredifferent.

By using such an apparatus, for example, in the pretreatment step, thespecimen is flowed through the support to which the antibody against thenonspecific reaction factor is immobilized or the support to which thereducing agent is immobilized by pumping. Therefore, corresponding tothe number of times of pumping the specimen, the number of times ofbeing flowed through the support to which the antibody against thenonspecific reaction factor is immobilized or the support to which thereducing agent is immobilized is increased, and the possibility that thenonspecific reaction factor may encounter the antibody against thenonspecific reaction factor or the reducing agent is increased.Therefore, when using the support to which the antibody against thenonspecific reaction factor is immobilized, the nonspecific reactionfactor can be bound to the antibody against the nonspecific reactionfactor more certainly to allow the nonspecific reaction factor to betrapped by the support, and when using the support to which the reducingagent is immobilized, the nonspecific reaction factor can be decomposedmore certainly. Further, as described above, the movement of the pistondriven at the time of pumping is controlled by the servomotor based onthe pressure detection signal from the pressure sensor. Therefore, theamount of the liquid sucked up at the time of pumping, the amount of theliquid discharged, the pressure of sucking up and the pressure ofdischarging can be controlled with high accuracy, and the flow of theliquid can be rapidly controlled. Therefore, trapping of the nonspecificreaction factor or decomposition of the nonspecific reaction factor asdescribed above can be carried out in a short time, and time requiredfor the pretreatment can be more reduced. Further, pumping is carriedout using a pipette chip and a well, and the sizes of the pipette chipand well may correspond to the sizes of the pipette chip and well usedin the labeling reaction step. By using the pipette chips of the samesize and the wells of the same size, the size of the assay system can bemore reduced. Note that the system explained above is just an example,which can be suitably changed.

When immunoassay is automatically performed using the assay system inthis way, by carrying out sucking up and discharging of the specimen inthe pretreatment with the tip portion of the pipette chip being immersedin the well, bubbling and spattering of the specimen can be reduced.Further, all of the step of the treatment of removing the contaminantusing the first support, the step of preparing the second support, thelabeling reaction step and the assay step are consistently carried outin limited places in the apparatus, for example, in the well and thepipette chip, or in the well, the pipette chip and the antigenseparation/immobilization tube. Therefore, the treatment process issimplified, and in addition, there is a high possibility that mixing ofbacteria and the like may be reduced and reduction of contamination canbe expected.

In the explanation above, the embodiment in which the nonspecificreaction factor is removed from the specimen is exemplified. However,the present invention is not limited thereto, and an antigen of interestmay be extracted from the specimen. Magnetic particles to which anantibody which specifically reacts with the antigen of interest isimmobilized are added to and suspended in the specimen. After thesuspension, the specimen is sucked up into the pipette chip and themagnet is moved toward the pipette chip to constrain the magneticparticles. After constraining the magnetic particles, the liquid in thepipette chip is discharged, and the antigen bound to the magneticparticles is washed, thereby removing the contaminant. Such apretreatment can also be carried out.

5-2. System for Assaying Nucleic Acid

In the explanation above, the assay system in which the pretreatmentstep of removing the nonspecific reaction factor from the specimen iscarried out before the immunoassay is exemplified, but the presentinvention can also be used for an assay system for nucleic acids. As theassay system for nucleic acids, for example, in the Stanford typesystem, on a microarray chip on which probe DNAs are arranged, a targetDNA solution is spotted to cause hybridization, and the microarray chipon which the target DNA solution is spotted is detected by a lightreceiving element or an image sensor to obtain detection data. In orderto more clarify a signal of the obtained detection data, it is necessaryto more certainly cause hybridization between the probe DNA and thetarget DNA. For certain hybridization between the probe DNA and thetarget DNA on the microarray chip, removal of the contaminant in thespecimen is one of important problems. The smaller the amount of thecontaminant mixed in the target DNA solution spotted on the microarraychip is, the higher the quality of the detection data obtained is. Thepresent invention can be used for this pretreatment of removing thecontaminant in the target DNA solution.

For example, for removing the contaminant contained in the target DNAsolution, the specimen containing the target DNA is held in a well, andmagnetic particles to which a substance having affinity to thecontaminant in the specimen is immobilized are added to the specimen inthe well using the pipette chip, followed by pumping.

After pumping, the specimen is sucked up into the pipette chip, and themagnet is moved toward the pipette chip to constrain the magneticparticles. By discharging the liquid into the well with the magneticparticles being constrained, the contaminant can be removed from thespecimen. By preparing the target DNA solution from the specimen fromwhich the contaminant has been removed, the contaminant contained in thetarget DNA solution can be reduced. Further, when the steps from thestep of preparing the target DNA solution to the step of obtaining thedetection data are consistently automated in one system, the treatmentprocess is simplified, and in addition, prevention of contamination andimprovement of convenience can be expected. In the explanation above,DNA is exemplified as a target, but this technique can also be used forpreparing other nucleic acid targets such as cRNA and mRNA.

EMBODIMENTS

As explained above, by changing the combination of the embodiment of thepretreatment step and the embodiment of the assay step, it is possibleto produce assay systems of various embodiments. Some examples ofpossible combinations of embodiments of the pretreatment step, thelabeling reaction step and the assay step will be described below.

Embodiment 1

An example in which CA19-9 that is utilized at the time of cancer testsfor the digestive system is used as an antigen will be described.Naturally, the present invention is not limited to this embodiment. FIG.23 is a flow chart of a case where a pretreatment is carried out usingmagnetic particles to which an antibody against a nonspecific reactionfactor is immobilized and subsequently an immunoassay is carried out. Asshown in FIG. 23, in the pretreatment of a specimen, a serum is used asthe specimen, and magnetic particles to which at least one of protein A(antibody against a nonspecific reaction factor) and protein G (antibodyagainst a nonspecific reaction factor) that can bind to globulin(nonspecific reaction factor) contained in the specimen is immobilized(hereinafter referred to as “magnetic particles for pretreatment”) areused as the magnetic particles to which the antibody against thenonspecific reaction factor is immobilized.

Firstly, the magnetic particles for pretreatment (first support) aremixed with the serum in a well, and sucking up and discharging themixture is repeated for suspending. After suspending, the suspension issucked up into a holding portion of a pipette chip, a magnetic field isprovided to the holding portion to constrain the magnetic particles atone position in the holding portion, and the remaining solution isdischarged into the well. In this way, the globulin contained in thespecimen can be trapped by the protein A or protein G of the magneticparticles for pretreatment and removed. When it is desired that theglobulin is removed with higher accuracy, this step of the treatment ofremoving the contaminant may be suitably repeated. Note that whenremoving the globulin and the like contained in the specimen with higheraccuracy, it is also effective, for example, to use a protein-removingagent such as an enzyme in combination.

Magnetic particles for specific reaction to the surface of which ananti-CA19-9 antibody is immobilized (second support) are mixed with andsuspended in the serum obtained by removing the globulin. The CA19-9antigen in the treated serum binds to the anti-CA19-9 antibody, andtherefore the CA19-9 antigen is trapped by the magnetic particles forspecific reaction. The suspension is sucked up into the holding portionof the pipette chip, a magnetic field is provided to the holding portionto constrain the antigen-bound magnetic particles at one position in theholding portion, and the remaining solution is discharged into the well.The antigen-bound magnetic particles, which are held in the holdingportion, and to which the CA19-9 antigen has been bound, are mixed witha washing solution held in another well and washing is performed. Afterwashing, the magnetic field is provided to the antigen-bound magneticparticles to which the CA19-9 antigen has been bound, and they areseparated from the washing solution. The antigen-bound magneticparticles separated from the washing solution are mixed with andsuspended in an enzyme labeling anti-CA19-9 antibody solution held inanother well. In this way, the CA19-9 antigen can form sandwich bindingwith the anti-CA19-9 antibody and the enzyme labeling anti-CA19-9antibody.

The magnetic field is provided to the labeled antibody-bound magneticparticles having the CA19-9 antigen that has been subjected to sandwichbinding, and the labeled antibody-bound magnetic particles are separatedfrom the suspension. The labeled antibody-bound magnetic particlesseparated are mixed with a washing solution in another well and washingis performed. After washing, the magnetic field is provided to thelabeled antibody-bound magnetic particles having the CA19-9 antigen thathas been subjected to sandwich binding, and the labeled antibody-boundmagnetic particles are separated from the washing solution. The labeledantibody-bound magnetic particles are mixed with and suspended in asubstrate solution held in another well. After the elapse of the enzymereaction time, the suspension is subjected to photometry to measureabsorbance, emission intensity, etc.

Embodiment 2

FIG. 24 is a flow chart of a case where a pretreatment is carried outusing a column to which an antibody against a nonspecific reactionfactor is immobilized and subsequently an immunoassay is carried out. Asshown in FIG. 24, like Embodiment 1, a serum is used as the specimen,and an affinity column (first support) to which at least one of proteinA and protein G that can bind to globulin contained in the specimen isimmobilized is used to treat the serum. The serum in a well is sucked upinto a column-containing pipette chip, and via an affinity column, theserum held in the pipette chip is discharged into the well. When suchsucking up and discharging of the serum are carried out a predeterminednumber of times, the globulin contained in the serum can be bound to theprotein A or protein G of the affinity gel and removed. After that, thetreated serum discharged into the well can be subjected to a reactionstep in a manner similar to that in Embodiment 1.

Embodiment 3

In this embodiment, the pretreatment is carried out by using any one ofthe methods of the treatment of removing the contaminant alreadydescribed in FIGS. 8-15, and the assay is carried out using the antigenseparation/immobilization tube shown in FIG. 21, wherein a plurality ofantigens are simultaneously bound to respective antibody-immobilizedbeads. After a certain period of time, a washing solution in anotherwell is sucked up to wash the antibody-immobilized beads. After washingthe antibody-immobilized beads, an enzyme labeling solution is sucked upinto the antigen separation/immobilization tube. After the enzymelabeling solution is sucked up into the tube and an enzyme labelingantibody is bound to each antigen, a washing solution in another well issucked up to wash the antibody-immobilized beads. After washing, asubstrate solution is sucked up into the antigenseparation/immobilization tube. After the elapse of sufficient time,absorbance, emission intensity, etc. of each of the antibody-immobilizedbeads which have been subjected to sandwich binding are measured.

In this way, by treating the specimen using theseparation/immobilization tube in which a plurality of types ofantibody-immobilized beads are arranged, a plurality of antigens can betrapped at a time. Therefore, the operation can be simplified, and inaddition, the time required for detecting the antigens in the specimencan be dramatically reduced.

In the present invention, the pretreatment may be carried out using anyone of the methods of the treatment of removing the contaminantexemplified in FIGS. 8-15, and the assay may be carried out byconventional ELISA after the pretreatment. For example, the treatment ofremoving the contaminant is carried out using any one of the methods ofthe treatment of removing the contaminant shown in FIGS. 8-15. Inanother well, the antibody against the antigen is immobilized inadvance, and when a serum from which the nonspecific reaction factor hasbeen removed by the treatment of removing the contaminant is added tothis well, the antigen in the treated specimen can bind to the antibodyin the well. After washing, a labeling solution containing an enzymelabeling antibody is added to this well, and the enzyme labelingantibody can bind to the antibody-bound antigen. After washing, asubstrate solution is added to cause color development, and achromogenic reaction terminating solution is suitably added. The well inwhich color development has been caused is set in an absorbancemeasurement device to measure absorbance, etc. An assay system forcarrying out such a process can also be realized.

Embodiment 4

In order to perform the process from the pretreatment of the specimen tothe detection of the substance of interest, in the present invention, aseries of steps including the pretreatment of the specimen may becarried out using a cartridge into which a holding portion for holdingthe specimen, a holding portion for holding the magnetic particles forremoving the contaminant from the specimen in advance, a holding portionfor holding the magnetic particles to which the antibody for labelingthe antigen in the specimen is bound in advance, a holding portion forholding the substrate solution in advance, etc. are integrated inadvance. Further, it is also possible to allow the assay system forspecimen using the cartridge to consistently carry out the step of thetreatment of removing the contaminant and the subsequent labelingreaction step or the assay step comprising the labeling reaction step.Such an assay system using the cartridge will be described below. FIG.25 is a schematic view of an assay system utilizing a cartridge in whichmagnetic particles for pretreatment and a substrate solution are held inadvance. FIG. 26 is an explanatory drawing for schematically explaininga mode of operation of the assay system utilizing the cartridge. Asshown in FIGS. 25 and 26, an assay system 150 has a magnet 151, adispensing apparatus 152, a heat block 153, a detection apparatus 154,an arrangement control apparatus, a central control apparatus, etc.

The dispensing apparatus 152 has a pump 160 and a nozzle 162, and on thenozzle 162, a pipette chip 164 can be detachably mounted. The detectionapparatus 154 has a photomultiplier tube (hereinafter referred to as“PMT”) 172 and a light source 170. The PMT 172 receives light from thelight source 170, and in response to this, a signal is outputted fromthe PMT 172. As shown in FIG. 26, for example, the nozzle 162, the PMT172 described below and the light source 170 are controlled by thecentral control apparatus to move in the vertical direction, and themagnet 151 and the cartridge 180 are controlled by the central controlapparatus to move in the horizontal direction.

The cartridge 180 has a base panel 181 that is formed into an elongateshape and a plurality of holding portions 182, and the plurality ofholding portions 182 are arranged from one end to the other end of thepanel 181 in the longitudinal direction. In the cartridge 180, the base181 formed on the base panel and the plurality of holding portions 182are integrally formed. Each holding portion 182 has an opening on thebase panel to allow the pipette chip 164 to be received.

As the holding portions 182 provided to the cartridge 180, a holdingportion for holding a solution of magnetic particles for pretreatment, aholding portion for holding a specimen, a holding portion for holding asolution of magnetic particles to which an antibody for labeling isbound, a holding portion for holding a washing solution for washing anantigen binding to the magnetic particles for labeling, a holdingportion for holding a substrate solution for causing a chromogenicreaction, etc. are provided. The types of the holding portions providedto the cartridge 180 are not limited to the examples described above.For example, holding portions may be provided to the cartridge to allowonly the pretreatment to be carried out.

A holding portion 182 a for measuring absorbance is provided to the basepanel 181 (for example, the central portion thereof), and on the upperside of the holding portion 182 a, a mount 184 for the PMT 172 to bemounted is positioned. The base panel 181 and the holding portions 182are protected from light by an aluminum seal or the like, and the PMT172 is fitted into the mount 184 in a state where it is shielded fromlight. The bottom portion of the holding portion 182 a for detection canbe fitted into the light source 170 in a state where it is shielded fromlight. Irradiation light from the light source 170 fitted to the bottomportion of the holding portion 182 a for detection is received by thePMT, thereby counting the number of photons.

The position of the cartridge 180 relative to the nozzle 162 and the PMT172 is determined by a cartridge controller (not shown). The cartridgecontroller controls the cartridge 180 to move in the horizontaldirection and determines the position of the cartridge 180 relative tothe dispensing apparatus 152 and the detection apparatus 154. Bycontrolling the position of the cartridge 180, for example, thecartridge can be appropriately located relative to the pipette chip, andtherefore, pipetting and measurement of absorbance in the step of thetreatment of removing the contaminant, the labeling reaction step andthe detection step can be smoothly carried out

The holding portions 182 of the cartridge 180 has, for example, a firstportion 187 for carrying out the treatment of removing the contaminantand a second portion 188 for carrying out the labeling reaction step andthe detection step. A holding portion to which a heater 153 can befitted is provided to the cartridge 180 in order to perform incubation,but this holding portion for the heater may be suitably omittedaccording to the purpose of the assay system.

Hereinafter, the action of the assay system 150 utilizing the cartridge180 will be described. After mounting the cartridge 180, a solution ofmagnetic particles held in a first holding portion of the cartridge 180in advance is sucked up into the pipette chip 164 and discharged into asecond holding portion, followed by pumping. FIG. 27 shows thepretreatment step in the assay system utilizing the cartridge. As shownin FIG. 27, after pumping, the liquid in the second holding portion issucked up into the pipette chip 164, the magnetic particles to which thecontaminant is bound are separated by the magnet 151, and an assaysample from which the contaminant has been removed is discharged into athird holding portion. In the third holding portion, a mixture of themagnetic particles for labeling and the assay sample is held, and thismixture is pumped and sucked up into the pipette chip. After sucking upthe mixture, the antigen-bound magnetic particles are separated by themagnet and put into a fourth holding portion. The antigen binding to themagnetic particles is washed with a washing solution held in the fourthholding portion in advance, and a mixture of the washing solution andthe antigen is sucked up into the pipette chip. From the mixture suckedup into the pipette chip, the antigen-bound magnetic particles areseparated by the magnet 151, and the antigen is put into a fifth holdingportion 182 a for detection.

FIG. 28 shows the assay step in the assay system utilizing thecartridge. As shown in FIG. 28, a substrate solution is held in thefifth holding portion 182 a in advance, and the substrate solution isreacted with the antigen to cause color development, thereby detectingabsorbance. When removing the contaminant in the specimen, in the casewhere it is desired to remove the contaminant contained in the specimenusing a membrane, affinity column or reducing agent-immobilized gel, apipette chip having a membrane, a pipette chip containing an affinitycolumn, or a pipette chip containing a reducing agent-immobilized gel isattached to the nozzle 162 to treat the specimen. For example, as shownin FIG. 28, when removing the contaminant contained in the specimenusing a pipette chip 190 having a membrane, the pipette chip 190 havingthe membrane is fitted to the nozzle 162 to treat the specimen. In thisway, it is possible to ensure variation of the technique of removing thecontaminant in the assay system 150, and convenience of the system canbe improved.

Thus, the holding portions that are required in a series of stepsincluding the step of pretreating the specimen are provided to thecartridge 180 in advance, and this cartridge 180 is mounted to the assaysystem 150 to be used. In this way, users can save effort to prepare asolution of magnetic particles, a washing solution, etc., andconvenience of the operation of the assay system 150 can be improved.

In the embodiment above, the pretreatment is carried out by moving thepipette chip in the vertical direction and moving the cartridge in thehorizontal direction, but the technique of carrying out the pretreatmentis not limited thereto. For example, the pretreatment of the specimenmay be carried out by fixing the cartridge and moving the pipette chipin the horizontal direction and vertical direction. By fixing thecartridge, designers can save effort to design the system so that theliquid in the well is not spattered to the outside, and it becomespossible to provide a more convenient assay system.

In the embodiment 4 above, the cartridge 180, in which the holdingportion 182 a in which the substrate solution is held in advance isprovided at the central portion and the holding portion to which theheater 153 for culturing can be fitted is provided at the end portion,is exemplified, but the arrangement order of the holding portions in thecartridge is not limited thereto. FIG. 29 is a schematic view of acartridge having another form. For example, as shown in FIG. 29, theform may be suitably changed depending on the structure of the assaysystem to which the cartridge is mounted. In the form shown in FIG. 29,a holding portion 202 a, to which a heater 153 can be fitted, isprovided at the central portion of a cartridge 200, and a holdingportion 202 b, in which a substrate solution is held in advance, isprovided at the end portion. By suitably changing the arrangement orderof the holding portions in this way in view of the content of thetreatment, the treatment of the specimen can be carried out moreefficiently.

Further, in the embodiment described above, the solution of magneticparticles is held in the holding portion 182 in advance, but it is alsopossible to use a pipette chip in which the solution of magneticparticles is held in advance or to use a pipette chip having a supportfor adsorbing the contaminant in the specimen in order to carry out thepretreatment to of the specimen. In this case, it is required tosuitably change the structure of the holding portions of the cartridgecorresponding to such a pipette chip.

Further, in the explanation above, the cartridge 180 in which the firstportion 187 and the second portion 180 are integrally formed isexemplified, but the structure may be such that the first portion 187and the second portion 188 can be freely combined. By providing thestructure in which the first portion 187 and the second portion 188 canbe freely combined, users can select any of the first portion, thesecond portion, and the combination of the first portion and the secondportion according to individual cases (the case where only the treatmentof removing the contaminant from the specimen is desired to be carriedout, the case where only the steps from the labeling reaction step tothe detection step (excluding the pretreatment step) are desired to becarried out, and the case where all the steps are desired to be carriedout), and it is possible to provide a more convenient assay system.

In the embodiment described above, CA19-9 is used as the antigentargeted for detection, but the detection target is not limited thereto,and examples thereof include simple protein such as rheumatoid factor,free thyroxine (F-T4), thyroid-stimulating hormone (TSH), insulin andα-fetal protein (AFP), complex protein, steroid hormone and peptidehormone.

6. Nucleic Acid Assay System

As another example of the present invention, an embodiment in which thepresent invention is used in the field of nucleic acid detection will bedescribed below. When a nucleic acid is extracted from a specimen andthe extracted nucleic acid is amplified to be assayed, as a firstsupport, a support to which a substance having affinity to the targetnucleic acid is immobilized can be used to extract the target nucleicacid from the specimen, and as a second support, a support to which areagent for detecting the target nucleic acid is immobilized can be usedto assay the target nucleic acid. In this assay system, before the stepof detecting the target nucleic acid in the specimen, the step ofextracting the nucleic acid using the first support is carried out.Moreover, the system of the present invention includes a step ofpreparing the second support to which a substance having affinity to abiologically-relevant substance and/or labeling thebiologically-relevant substance is immobilized. In one embodiment of thepresent invention, the pretreatment including the step of extracting thenucleic acid using the first support and the step of preparing thesecond support is consistently carried out in one system. Hereinafter,the step of detecting the nucleic acid using the present invention willbe described.

FIG. 30 is an explanatory drawing for schematically showing thepretreatment step from extraction of a nucleic acid from a specimen topreparation of a second support and the detection step. As shown in FIG.30, in the system for assaying a target nucleic acid, (i) a step ofextracting the target nucleic acid from the specimen using a support(first support) to which a substance having affinity to the targetnucleic acid is immobilized, (ii) a step of preparing a support (secondsupport) which has at least one of the function of having affinity to abiologically-relevant substance and the function of labeling thebiologically-relevant substance, and (iii) a step of labeling anddetecting the target nucleic acid using the prepared second support arecarried out, and preferably the pretreatments (i) to (iii) areconsistently carried out. The specimen is held in a well 250 provided tothe assay system by a user.

6-1. Step of Extracting Target Nucleic Acid from Specimen

In order to obtain the target nucleic acid, a pipette chip (alsoreferred to as “dispensing chip”) having magnetic particles as the firstsupport to which a probe for trapping the nucleic acid is immobilized(hereinafter referred to as “magnetic particles for trapping nucleicacid”) is fitted to a nozzle. The specimen held in the well is sucked upinto the pipette chip and mixed with the magnetic particles for trappingnucleic acid. After mixing, the target nucleic acid specifically bindsto the probe immobilized to the magnetic particles for trapping nucleicacid and is trapped by the magnetic particles for trapping nucleic acid.

A magnet is moved toward the pipette chip with the magnetic particlesfor trapping nucleic acid to which the target nucleic acid binds beingsucked up into the pipette chip, and the magnetic particles for trappingnucleic acid are constrained in the pipette chip. The pipette chip inwhich the magnetic particles for trapping nucleic acid are constrainedby the magnet is moved away from the well and moved to a well holding awashing solution, and the magnet is moved away, thereby releasing themagnetic particles for trapping nucleic acid into the washing solution.The magnetic particles for trapping nucleic acid are washed with thewashing solution, and after that, a reagent or the like is added theretoto separate the target nucleic acid from the magnetic particles, therebyobtaining the target nucleic acid.

6-2. Step of Preparing Support for Detecting Target Nucleic AcidObtained

Meanwhile, in order to detect the extracted target nucleic acid, asupport to which a substance having affinity to a biologically-relevantsubstance and labeling the biologically-relevant substance isimmobilized is prepared. In the detection of the target nucleic acid,the target nucleic acid is labeled and amplified. As a labeling methodand an amplification method, a publicly-known method such as PCR method,PT-PCR method, real-time PCR method, LAMP method, RT-LAMP method, ICANmethod, SDA method, RCA method and NASBA method can be used. There arevarious techniques for performing the real-time PCR method, and forexample, an intercalation method, a hybridization method, a LUX methodor the like can be used.

For labeling and amplifying the nucleic acid, a solid-phased mastermixture (MMX) containing a probe or primer for labeling and amplifyingthe nucleic acid can be used. When using the real-time PCR method, it ispreferred that a primer or probe for amplifying the nucleic acid issuitably selected depending on the type of the nucleic acid to beamplified. As a probe or primer, for example, various products such as aTaqMan (registered trademark) probe, a FRET probe and a LUX primer canbe used. The probe has a label for labeling the target nucleic acid.This probe having the label is allowed to hybridize to the targetnucleic acid, thereby labeling the target nucleic acid. The primer andprobe can be suitably designed depending on the type of the nucleic acidtargeted for the detection. For example, when the hybridization methodis used as the real-time PCR method, the target nucleic acid issubjected to thermal denaturation, annealing and elongation reaction tolabel the target nucleic acid.

In the present invention, before carrying out the detection of thetarget nucleic acid, a reagent to be used for amplification of thenucleic acid is prepared in advance, and the reagent is solid-phased andheld in a well. In this way, a well (second support) to which a reagentthat has been solid-phased (hereinafter referred to as “solid-phasedreagent”) is immobilized is prepared. At the time of amplification ofthe nucleic acid, a buffer solution, the nucleic acid, etc. are providedto the solid-phased reagent, and then labeling and amplification of thetarget nucleic acid is started. By using a large volume of mastermixture, at the time of the assay step, it is possible to dispense itinto wells for holding an assay sample, and work efficiency can beimproved. The method for solid-phasing the reagent is not particularlylimited, but when the reagent is freeze-dried, convenience is improved,and improvement of work efficiency can be expected.

When the reagent of the master mixture is freeze-dried, for example, theprimer and probe for amplifying the target nucleic acid, and aprotecting/stabilizing agent such as saccharide and polyvinylpyrrolidonefor protecting and stabilizing the primer and probe are mixed together,and the mixture is cooled at a predetermined temperature and thepressure is reduced. The pressure at the time of preparation, coolingtemperature and cooling time may be suitably changed depending on theproperty of the master mixture of interest. Further, in the presentinvention, it is preferred that sucrose, lactose, trehalose or the likeis mixed so that a freeze-dried product is immobilized to the inside ofa container as a solid phase (as the second support) when freeze-dried.In this way, for example, a freeze-dried reagent can be immobilized tothe inside wall of a container, providing a film-like layer.

6-3. Step of Detecting Target Nucleic Acid

The detection of the target nucleic acid is carried out by putting thetarget nucleic acid into a well having the solid-phased reagent. Afterputting the target nucleic acid into the well, the target nucleic acidhybridizes to the probe contained in the solid-phased reagent, and thenthe detection becomes possible. For example, by measuring luminescence,fluorescence or the like, the target nucleic acid can be detected. Theimage data of the target nucleic acid can be obtained by using, forexample, a fluorescence laser microscope having an image sensor as adetection device. Fluorescence intensity, etc. can be calculated bysuitably analyzing the image data. By calculating the fluorescenceintensity in this way, the target nucleic acid can be detected.

Next, a system for automatically carrying out each of theabove-described steps will be described. An assay system forautomatically carrying out the above-described procedure has an assaysample-obtaining apparatus for holding a specimen and obtaining a targetnucleic acid and a detection apparatus for amplifying and detecting theobtained assay sample using the PCR method or the like.

The assay sample-obtaining apparatus has, for example, a well forholding a specimen and a well for holding a reagent. In the well forholding a specimen, a specimen obtained by a user such as a tissue, acell and a body fluid is held. When the specimen is a tissue or a cellfragment, it is preferably minced in advance.

The well for holding a reagent has: a plurality of wells; a pipettechip; a reagent for dissolving the specimen; magnetic particles, whichare put into the dissolved specimen, and to which a probe specificallybinding to a target nucleic acid is immobilized; a magnet forconstraining the magnetic particles in the pipette chip; an elutingreagent for separating and eluting the target nucleic acid from themagnetic particles which specifically bind to the nucleic acid via theprobe; etc. The target nucleic acid such as a DNA and an RNA isextracted from the specimen held in the well for holding a specimen.

The well for holding a reagent further has a well for holding asolid-phased reagent for labeling and amplifying the target nucleic acidextracted from the specimen. In the well, for example, a buffer, aprimer, a probe, nucleic acid polymerase, a distilled water, a washingsolution and the like are solid-phased and held. The solid-phasedreagent can be prepared, for example, by mixing respective reagentstogether and freeze-drying the mixture (freeze-dried master mixture).

The target nucleic acid can be extracted as follows: the specimen isdissolved, and the obtained solution is mixed with the magneticparticles to which the probe specifically binding to the target nucleicacid is immobilized; after mixing, the magnet is used to constrain andseparate the target nucleic acid-bound magnetic particles in the pipettechip; and the separated magnetic particles are washed, followed byeluting the target nucleic acid.

The detection apparatus carries out the action of amplifying anddetecting the target nucleic acid extracted from the specimen. As thetechnique of amplifying the nucleic acid, a real-time PCR method isexemplified. Amplification of the target nucleic acid can be carried outusing the freeze-dried master mixture held in the well for holding areagent according to the real-time PCR method. The detection apparatushas a reaction container for nucleic acid amplification, a temperatureadjustment portion for adjusting the temperature of the reactioncontainer, etc., and respective steps of thermal denaturation, annealingand elongation reaction of the nucleic acid can be repeatedly carriedout. After amplification of the target nucleic acid, the labeled targetnucleic acid is irradiated, for example, with an electromagnetic wavefor excitation, or a substrate solution for fluorescence reaction isadded and then the target nucleic acid is scanned by a scanner or thelike, thereby detecting the target nucleic acid.

Hereinafter, a more specific example of the above-described system willbe described. FIG. 31 is a top view of wells for carrying out fromextraction to detection of the target nucleic acid. As shown in FIG. 31,the assay system 480 for the target nucleic acid has treatment lines.FIG. 31 exemplifies 12 treatment lines from a first treatment line 500Ato a twelfth treatment line 500L. In the respective treatment lines 500Ato 500L, for example, a well 502 for holding a specimen; a well 504 forholding a dissolving solution; a well 506 for holding a buffer solution;a well 508 for holding magnetic particles; wells 510 for holding awashing solution for washing the nucleic acid extracted from thespecimen and a washing solution for washing a pipette chip; a well 512for holding an eluting solution for separating the target nucleic acidfrom the magnetic particles; a well 514 for temporarily holding an assaysample in which the nucleic acid has been extracted from the specimen;and wells 516 for holding a solid-phased master mixture for labeling theassay sample and detecting the target nucleic acid are arranged.

Above the first to twelfth treatment lines 500A to 500L, 12 nozzlescorresponding to the respective treatment lines 500A to 500L are movablyprovided in the line direction P (not shown), and to each nozzle, apipette chip is suitably fitted. The assay system 480 has, for example,a shielding door for isolating a well-arranged space from the outside,and the wells provided to the first to twelfth treatment lines can beshielded from the outside by this shielding door. In the explanationabove, the apparatus having the 12 treatment lines is exemplified, butthe number of treatment lines is not limited thereto. For example, thenumber of treatment lines may be 1 or 2, or 20 or 30 in order toincrease processing ability.

As shown in FIG. 32, the assay system 480 has a central processing unit532, a chip position controller 534, a chip mount controller 536, amagnetic field controller 538, a PCR unit 540, a pumping controller 542,a detector 545, a RAM 548, a ROM 550, a display panel 552, an operationinterface 554, a timer, etc.

The chip position controller 534 has mutually orthogonal axes X, Y andZ, and the position of the nozzle is controlled by a stepping motor or aservomotor. Regarding the axes X, Y and Z, for example, the axis X isapproximately parallel to the well arrangement direction of thetreatment lines, the axis Y is approximately perpendicular to the axis Xand approximately parallel to the direction of traversing the lines, andthe axis Z is approximately perpendicular to a plane made by the axis Xand the axis Y. When the treatment of the specimen is started and eachnozzle is moved, for example, the nozzle is driven in two steps, i.e., amovement on the axis X and a movement on the axis Z, thereby carryingout the treatments along the respective treatment lines 500A to 500L.

The chip mount controller 536 performs attachment of the pipette chip tothe nozzle and detachment of the pipette chip from the nozzle. The chipmount controller 536 has a gripping portion for gripping the pipettechip and a chip preparation portion for preparing another new pipettechip. When the nozzle is moved upward along the axis Z with the pipettechip being gripped by the gripping portion, the pipette chip is detachedfrom the nozzle. Next, the bared nozzle is moved on the axes X and Y tomove to a position above a new pipette chip. At the chip preparationportion, the new pipette chip is held with a mount portion side up and atip portion side down. When the nozzle is moved downward along the axisZ, the mount portion of the new pipette chip is attached to the nozzle.

The pumping controller 542 has a pump 580 and a pressure sensor 582, andcontrols sucking up and discharging of the liquid performed via thenozzle and the pipette chip attached to the nozzle. The pump 580 has ahousing formed into a cylindrical shape, a piston that is movably fittedinto the housing and a motor for driving the piston. The inside of thehousing communicates with the opening of the nozzle. The movement of thepiston is controlled, for example, by a servomotor, and driving of theservomotor is controlled by a drive control signal from the pumpingcontroller 542. When the piston is activated, it becomes possible tosuck up or discharge the liquid through the opening of the nozzle.

In the opening of the nozzle, a pressure sensor 582 for detecting thepressure is provided, and the pressure sensor 582 transmits a pressuresignal to the pumping controller 542. The pumping controller 542monitors the pressure based on the pressure signal from the pressuresensor 582. In this constitution, for example, when the tip portion ofthe pipette chip is immersed in the specimen in the well, the pressuredetected by the pumping controller 542 exceeds a predeterminedthreshold, and in response to this, the drive control signal istransmitted to the servomotor. Also at the time of sucking up anddischarging a fluid, the pressure sensor 582 constantly transmits thepressure signal to the pumping controller 542. Therefore, the pumpingcontroller 542 can control driving of the servomotor with high accuracy.In this constitution, the nozzle to which the pipette chip is attachedcan carry out sucking up and discharging the fluid, and the fluid can bestirred thereby.

In the ROM 550, various control programs are stored. According to a modeselected by a user via the operation interface 554, a control programread from the ROM 550 is developed to the RAM 548, and the centralprocessing unit 532 controls each portion of the assay system 480 basedon the control program developed in the RAM 548.

Examples of treatment programs to be stored in the ROM 550 include: (1)a first program for extracting an RNA from a cell or virus and detectinga PCR product after a PCR reaction; (2) a second program for extractinga DNA from a biological sample such as blood and detecting a PCR productafter a PCR reaction; and (3) a third program for extracting a plasmidDNA from a bacterium such as E. coli.

The display panel 552 displays items required to be provided to theuser. For example, the display panel 552 can display the number of timesof pumping at the time of extraction of nucleic acid, time to be allowedto stand after suspension of the magnetic particles, the flow rate atthe time of pumping, the amount to be sucked up and discharged, the rateof movement of the pipette chip, etc., and the user can confirm theseitems by the display. When set contents are desired to be changed, theycan be changed by operation of the operation interface 554.

The timer carries out timing according to a program read from the ROM550. Timing is carried out, for example, when pumping or thermaldenaturation, annealing and elongation reaction in a PCR reaction areperformed. By timing, a period of carrying out each step is accuratelymanaged.

The magnetic field controller 538 manages the placement of the magnet560 to control the strength of the magnetic field provided to thepipette chip. The magnetic field controller 538 has mutually orthogonalaxes X, Y and Z, and the placement of the magnet 560 is determined by astepping motor or a servomotor. The axes X and Y are approximatelyparallel to a plane in which wells are arranged and mutually orthogonal,and the axis Z is approximately perpendicular to the plane. At the timeof the movement of the magnet 560, for example, the placement of themagnet 560 can be determined in two steps, i.e., a movement on the axesX and Y and a movement on the axis Z.

The PCR unit 540 has a thermal sensor 570, a temperature controller 572and a heater 574. The temperature controller 572 detects a temperaturebased on a temperature signal from the thermal sensor 570. The thermalsensor 570 is located, for example, adjacent to a well 516 holding amaster mixture, and transmits the temperature signal to the temperaturecontroller depending on the temperature of the fluid in the well. Theheater 574 is located adjacent to the well 516, and energization of theheater 574 is controlled by the temperature controller 572. Thetemperature controller 572 controls energization of the heater 574 basedon the temperature signal from the thermal sensor 570, therebycontrolling the temperature of the fluid in the well 516. In this way, aPCR reaction, which requires appropriate temperature control, can berapidly carried out. The cycle of PCR reaction is basically constitutedby repeat of a thermal denaturation step, an annealing step and anelongation reaction step, and the temperature controller 572 controlsthe temperature of the fluid using the heater 574 so that thetemperature becomes optimum in each step. The data regarding the optimumtemperature, reaction time and the number of cycles of reaction in PCRreaction are stored in ROM in advance and read and carried out inresponse to a treatment mode selected by the user.

The detector 545 has: a trigger light source 590; a light guidingportion 592 for sending a trigger light from the trigger light source tothe liquid in the well; a light receiving portion 594 for receivinglight from the nucleic acid fluorescing due to light sent from the lightguiding portion 592; a detection circuit 596; etc. The light guidingportion 592 can be constituted, for example, by using optical fibers,and can guide the trigger light from the trigger light source in theoptical fibers to send the trigger light to the liquid in the well. Forthe light receiving portion 594, for example, image sensors such as CCDand MOS can be used, and the light receiving portion 594 receives lightfrom the fluorescing nucleic acid to output a light receiving signal tothe detection circuit 596. The detection circuit 596 detects the nucleicacid based on the light receiving signal from the light receivingportion 594.

The action of the nucleic acid detection system of the present inventionwill be described using a flow chart of FIG. 33. The treatment programselected by the user is read from the ROM 550, and based on the readtreatment program, the action of each portion of the assay system 480 isstarted. By the user, the obtained specimen is manually put into thewell 502 of each treatment line (the first to twelfth treatment lines500A to 500L in FIG. 31), and by the shielding door, the first totwelfth treatment lines 500A to 500L are shielded from the outside.After shielding of the first to twelfth treatment lines 500A to 500L isdetected, the treatment of the specimen is started, and the 12 nozzlesand pipette chips provided corresponding to the first to twelfthtreatment lines 500A to 500L are driven, thereby mixing the specimenwith the dissolving solution. After stirring of the mixture, themagnetic particles are added to the mixture, followed by stirring.

After the mixture to which the magnetic particles have been added issufficiently stirred, using the magnet, the magnetic particles areconstrained in the pipette chip to discharge unnecessary liquid to theoutside of the pipette, thereby obtaining the magnetic particles towhich the target nucleic acid has bound. The obtained magnetic particlesare discharged into the well holding the washing solution and washed.After washing, the magnetic particles are mixed with a separatingsolution for breaking the bond with the target nucleic acid and themixture is stirred. After the separating solution to which the magneticparticles have been added is sufficiently stirred, the magneticparticles are constrained in the pipette chip to separate the targetnucleic acid. The obtained fluid containing the target nucleic acid, asthe assay sample to be provided to the assay step, is held in the well514. The assay sample held in the well 514 is dispensed into the well516 in which the master mixture is held in advance to perform PCRreaction. In the PCR reaction step, the thermal denaturation step, theannealing step and the elongation reaction step are repeated apredetermined number of times to produce the PCR product based on thetarget nucleic acid. After carried out a predetermined number of times,the PCR product is detected by the detector 545.

Thus, by providing the 12 treatment lines typified by the first totwelfth treatment lines 500A to 500L, 12 specimens can be can besimultaneously treated, and therefore the efficiency of the treatment ofspecimen can be improved. Further, by arranging the well 502 for holdingthe specimen, the well holding the magnetic particles for target nucleicacid extraction, the well for washing and the well holding the mastermixture in a line, the nozzle and the pipette chip can be driven withoutloss, and treatment time can be further shortened. Moreover, since thenozzle and the pipette chip are driven in a linear fashion, mixing ofthe specimen of another line can be prevented, and contamination can bereduced.

In the explanation above, the assay system having the 12 treatment linesis exemplified, but the number of treatment lines is not limitedthereto, and the number may be more or less than that. Further, in theembodiment above, the assay system having the 12 treatment lines isexemplified, but other than the treatment in a linear fashion, forexample, a block for holding the specimen, a block for reacting thespecimen with the magnetic particles, a block for holding the washingsolution for washing the magnetic particles, etc., a block for holdingthe prepared assay sample, a block for PCR reaction, a block forassaying the amplified target nucleic acid and the like may be arrangedin a circular pattern or in a cross shape.

Further, the embodiment of the system is not limited to that describedabove and can be suitably changed. FIG. 34 is a schematic view of anassay system in which a nucleic acid can be pretreated utilizing acartridge. For example, as shown in FIG. 34, a target nucleic acid canbe detected using a cartridge in which a specimen holding portion and areagent holding portion are integrated. Hereinafter, the system fortreating the specimen using the cartridge in which the specimen holdingportion and the reagent holding portion are integrated will bedescribed.

An assay system 260 has a system body 262 and a cartridge 264 to beloaded on the system body 262. The system body 262 has: a rack 267 forholding the cartridge 264; a movement control mechanism 270 forcontrolling the movement of the rack 267 between the drawn position atwhich the rack is drawn out from the system body 262 and the housedposition at which the rack is housed in the system body; a pipette chiphaving the magnetic particles for obtaining the nucleic acid; aplacement control mechanism 273 for controlling the placement of thepipette chip relative to the cartridge 264; a chip attachment/detachmentcontroller 275 for attaching or detaching the pipette chip; a scanner277 for scanning and detecting the amplified nucleic acid; etc. The rack267 has a lid 280 for shielding the cartridge 264 positioned at thehoused position from the outside, and this reduces the risk of adherenceof bacteria to the cartridge 264 positioned at the housed position, etc.

Regarding the arrangement form of wells of the cartridge 264, thenumber, order, size, etc. of wells vary depending on whether a DNA or aRNA is extracted for amplification. FIG. 35 shows a cartridge to be usedin the above-described assay system, and is a perspective crosssectional view of wells holding the master mixture, a portion of whichis taken along the longitudinal direction of the cartridge. For example,in the case where a DNA is extracted and amplified using the PCR method,as shown in FIG. 35, into the cartridge 264, a well 300 for holding thespecimen; a well 302 in which an extraction reagent for extracting DNAfrom the specimen is held in advance; a well 304 for holding a buffer;wells 306 and 308 for holding a washing solution for washing theextracted DNA; an eluting solution 310 for separating the DNA from themagnetic particles for target nucleic acid extraction to obtain the DNA;a well 312 for holding a washing solution for washing a pipette chip330; wells 314 for holding a freeze-dried master mixture; wells 316 forholding a substrate solution used when allowing the labeled DNA tofluoresce to be detected; etc. are integrated. The opening of each wellis sealed with an aluminum seal (not shown) so that bacteria do notinvade the inside of each well prior to use. In the wells 314, afreeze-dried master mixture 318 is provided to the inside wall of thecontainer, providing a film-like layer. As a technique of providing thefreeze-dried master mixture 318 in the film-like form in the wells 314,for example, a master mixture before freeze-dried is held in the wells314, and after that, freeze-drying is carried out under predeterminedfreeze-drying conditions, thereby providing the film-like freeze-driedmaster mixture 318 in the wells 314.

After the cartridge 264 is loaded on the rack 267 and automaticdetection of nucleic acid is started, the rack 267 is housed in thesystem body 262, and the cartridge 264 is isolated from the outside.After the cartridge 264 is positioned at the housed position by the rack267, the aluminum seal is detached, and extraction of DNA from thespecimen is carried out. To the cartridge 264, the wells 314 having thefreeze-dried master mixture are provided in advance, and therefore,after the extraction of DNA, the pretreatment can be rapidly finished,and it is possible to shift to the next assay step.

Thus, since it is possible to automatically and consistently carry outthe pretreatment using the cartridge 264, in the present invention, asimple and convenient system can be provided. Further, by extracting thetarget nucleic acid from the specimen using the magnetic particles, theamount of unnecessary substances is reduced before amplification of thenucleic acid, thereby carrying out more reliable DNA detection.

Further, in the explanation above, the steps from the step of extractingthe target nucleic acid from the specimen to prepare the assay sample tothe step of detecting the target nucleic acid based on the preparedassay sample are consistently carried out, but it is also possible toindependently carry out the step of extracting the target nucleic acidfrom the specimen to prepare the assay sample and the assay step ofassaying the target nucleic acid based on the assay sample usingdifferent apparatuses. As one example of such a system, an assay system,which has: a sample preparation apparatus, wherein the specimen is held,the target nucleic acid is extracted and the assay sample is obtained;and a detection apparatus, wherein the obtained assay sample isamplified using the nucleic acid amplification method and detection iscarried out, is exemplified. When using such a system, as shown in FIG.36, in a well cartridge 600, a separation unit 615 into which a well 610for holding the assay sample and the wells 612 for holding the mastermixture are integrated can be split and separated from a cartridge body608, for example, along the splitting line L, and the separation unit615 is loaded on the detection apparatus, thereby carrying out thedetection of the target nucleic acid.

When the obtained specimen is held in the well cartridge 600 and it isloaded on a sample preparation apparatus, the treatment of the specimenis started, and the assay sample is held in the well 610 for holding theassay sample. When the separation unit 615 is separated from thecartridge body 608 and loaded on the detection apparatus, the assaysample held in the well 610 is poured into the 4 continuous wells 612holding the master mixture. After the assay sample is mixed with themaster mixture, the target nucleic acid is amplified according to thePCR method and then detected.

Thus, since the separation unit 615 into which the well 610 for holdingthe assay sample and the wells 612 for holding the master mixture areintegrated can be separated from the cartridge body 608, the step untilobtaining the assay sample and the step of detecting the target nucleicacid from the obtained assay sample can be respectively carried out indifferent apparatuses.

Therefore, the drive schedule of the sample preparation apparatus isindependent from the drive schedule of the detection apparatus.Accordingly, after preparation of a first assay sample, preparation of asecond assay sample can be immediately started, and therefore, the assaysample can be prepared more freely without the necessity of waiting thedetection of the target nucleic acid. In addition, it is possible totemporarily store the assay sample.

Further, in the explanation above, one specimen corresponds to one lineof the group of wells, but one specimen may also correspond to aplurality of lines of the group of wells. For example, as shown in FIG.37, one specimen can correspond to two lines of wells. An assay system620 has: a nucleic acid extraction portion 625 having wells to be usedat the time of extraction of nucleic acid; a solid-phased reagentholding portion 630 having wells holding a solid-phased reagent fornucleic acid amplification; a temperature adjustment portion 635 whichis combined with a thermal cycler; a detector 640 for sending a triggerlight and detecting a nucleic acid; a nozzle unit 645; etc. The nozzleunit 645 is driven within a work area 645 and carries out sucking up anddischarging the specimen. The nucleic acid may be assayed using thearrangement of wells in this way.

Further, in a preferred embodiment of the present invention, using theassay system described above, various viruses such as influenza viruses(e.g., H1N1, H3N2, H5N1, H7N7) can be detected. The detection ofinfluenza virus is carried out in the following order: extraction of aninfluenza virus from a specimen collected (e.g., body fluid in the nasalcavity); preparation of a master mixture; performing real-time RT-PCR;and detection.

[Probe and Primer for Detection of Influenza Virus a (H1N1)]

Examples of probes and primers to be used include:

Forward primers (InfA, SW InfA, SW H1, RnaseP)

Reverse primers (InfA, SW InfA, SW H1, RnaseP)

Taq Man probes (InfA, SW InfA, SW H1, RnaseP)

Therefore, combinations of a primer and a probe are as follows:

InfA: influenza A primer set and Taq Man probe

SW InfA: SW InfA primer set and Taq Man probe

SW H1: SW H1 primer set and Taq Man probe

RnaseP: human RNaseP gene (internal positive control) primer set and TaqMan probe

Mater mixtures, which contain the above-described 4 types ofcombinations of a primer and a probe, respectively, are prepared, andfor example, they are put in the 4 continuous wells 314 of the cartridge264 shown in FIG. 35 in advance, and the wells are sealed with analuminum seal. In this way, it is possible to provide a system by whichinfluenza virus A (H1N1) can be detected by simple operation, and acartridge for this system.

In the above-described example, the cartridge 264 carries out from thestep of extraction of nucleic acid to the step prior to the detectionregarding one specimen, but it is also possible to arrange a pluralityof cartridges 264 in parallel to allow simultaneous treatment of aplurality of specimens. By treating a plurality of specimenssimultaneously, it is possible to improve treatment capacity and toprovide a more convenient system.

In FIGS. 31-37 described above, the embodiment using the PCR method inwhich nucleic acid amplification is performed by increasing/decreasingthe temperature of a mixing solution of the target nucleic acid and thereagent for PCR reaction is exemplified, but the present invention isnot limited thereto, and it is also possible to use an isothermalamplification method in which the nucleic acid is isothermallyamplified.

The nucleic acid amplification apparatus of the present invention ischaracterized in that it comprises:

(a) a specimen holding portion in which a specimen is held;

(b) a first holding portion in which trapping particles for trapping atarget nucleic acid from the specimen are held;

(c) a second holding portion in which a reagent for detecting the targetnucleic acid is held;

(d) a dispensing mechanism for dispensing the specimen into the specimenholding portion, a mechanism for mixing the specimen with the trappingparticles to extract the target nucleic acid from the specimen, and amechanism for mixing the extracted target nucleic acid with the reagentfor detecting; and(e) a mechanism selected from the group consisting of: a mechanism forpouring a hydrophobic fluid, which has a specific gravity smaller thanthat of a mixed fluid of the target nucleic acid and the reagent fordetecting, into the second holding portion; a mechanism for removing orputting lids for covering the respective holding portions; a mechanismfor irradiating an irradiating light for letting the target nucleic acidfluoresce; a mechanism for receiving a light from the target nucleicacid irradiated with the irradiating light to detect the target nucleicacid; and a mechanism in which the mechanisms are combined.By changing the combination of the constitutions of (a) to (e) above,various types of apparatus can be realized.

Hereinafter, an apparatus having a mechanism of isothermally amplifyinga target nucleic acid will be described. Regarding the same points asthose for the above-described apparatus for amplifying a target nucleicacid using the PCR method, the outline thereof will be described, anddetailed description thereof is omitted.

FIG. 38 is a perspective view showing a plurality of wells and nozzlesfor carrying out from extraction to detection of a target nucleic acid.As shown in FIG. 38, in each of treatment lines 700A to 700F integratedinto a cartridge, for example, the following wells are arranged: a well702 for holding a specimen; wells 704 for holding a dissolving solution;wells 706 for holding a buffer solution; a well 708 for holding magneticparticles as trapping particles; a well 710 for holding a washingsolution for washing a nucleic acid extracted from the specimen or awashing solution for washing a pipette chip; a well 712 for holding aneluting solution containing a reagent for separating the target nucleicacid from the magnetic particles; a well 714 for temporality holding atarget nucleic acid-containing solution obtained after extraction ofnucleic acid from the specimen; wells 716 containing a dried reagent(e.g., freeze-dried reagent) for amplifying the target nucleic acid(reagent for detection); and a detection well 718 for detecting anamplified product obtained by amplifying the target nucleic acid in thetarget nucleic acid-containing solution.

Above the first to sixth treatment lines 700A to 700F, 6 nozzle units720 corresponding to the respective treatment lines 700A to 700F aremovably provided in the line direction P, and to each nozzle unit 720, apipette chip 730 can be fitted. The number of treatment lines is notlimited to 6 and can be suitably changed.

FIG. 39 is a partial perspective view showing the tip portion of thenozzle unit 720. As shown in FIG. 39( a), for example, the nozzle unit720 has: a pumping opening 730 for sucking up/discharging a fluid suchas a solution containing a specimen or target nucleic acid; a plasticoptical fiber (hereinafter referred to as “POF”) 732 for sending atrigger light for fluorescence reaction to an amplified product of thetarget nucleic acid; and a lens 734 for receiving a light from thetarget nucleic acid. The lens 734 is placed in the pumping opening 730,and around the pumping opening 730, for example, 8 POFs 732 are arrangedat equally-spaced intervals. The form of the nozzle unit is not limitedto the above-described one, and for example, nozzle units shown in FIGS.39( b) and (c) may also be employed. As shown in FIG. 39( b), to thecentral portion of the tip of a nozzle unit 760, a pumping opening 762for sucking up/discharging a fluid and a lens 764 are provided, and tothe surrounding portion thereof, 4 POFs 767 for sending a trigger lightare provided. Further, as shown in FIG. 39( c), it is also possible toemploy a constitution in which a pumping opening 772 and a POF 774 forsending a trigger light are provided to the central portion of the tipportion of a nozzle unit 770.

FIG. 40 is a cross sectional view of a nozzle unit 720 taken along aplane parallel to the drawing direction of a pumping opening 730. Asshown in FIG. 40, around the pumping opening 730, for example, 8 POFsare arranged, and in the center in the pumping opening 730, a lens 734and a POF 740 for transmitting optical images are placed. The lens 740is opposed to an amplified product-containing solution held in a well748 for detection, and provides an optical image of the amplifiedproduct-containing solution to the POF 740 side. The optical imageprovided by the lens 740 is sent through the POF 740 and input to animage reproduction optical system described below (see FIG. 41). Byarranging POFs 732 for sending a trigger light around the lens 734,shading in which the peripheral part of the image becomes dark issuppressed, and it is possible to obtain a high-quality image of anamplified product. Further, the structure of the tip portion of thenozzle unit is not limited to the above-described one, and it is alsopossible to employ the structure shown in FIG. 40( b). As shown in FIG.40( b), a nozzle unit 780 has a pumping opening 782 and a POF 784 forsending a trigger light. When a nozzle unit into which a pumpingopening, an optical fiber for sending a trigger light and/or an opticalfiber for sending an optical image of the inside of a well for aspecimen to an image sensor are integrated, and treatment lines linearlyarranged are used in this way, by only linearly moving the nozzle unitthat carries out both pumping of a specimen or the like and detection ofan amplified product of a target nucleic acid along the treatment line,it is possible to carry out from extraction of the target nucleic acidfrom the specimen to detection of the amplified product, and sizereduction in the apparatus can be expected. In particular, the diameterof the nozzle unit can be decreased by the pumping opening and theoptical fibers, and therefore the distance between adjacent nozzle unitsand the distance between treatment lines can be decreased. As a result,it is possible to further reduce the size of the nucleic acid detectionapparatus.

FIG. 41 is a functional block diagram of a target nucleic acid detectionapparatus. As shown in FIG. 41, an assay system 680 has a centralprocessing unit 832, a nozzle position controller 834, a chip mountcontroller 836, a magnetic field controller 838, an isothermal controlunit 840, a pumping controller 842, a timer 843, a detector 845, a RAM848, a ROM 850, a display panel 852, an operation interface 854, animage reproduction optical system 856, etc.

The nozzle position controller 834 has mutually orthogonal axes X and Z(2 axes), and the position of the nozzle unit 720 is controlled by 2motors, i.e., first and second motors 861 and 862. Regarding the axes Xand Z, for example, the axis X toward the direction P is approximatelyparallel to the arrangement direction of wells in each of the treatmentlines 700A to 700F, and the axis Z is approximately perpendicular to theaxis X and approximately parallel to the direction of the line betweenthe proximal position and the distal position of wells. When thetreatment of the specimen is started and each nozzle unit 720 is moved,for example, the nozzle 720 is driven in two steps, i.e., a movement onthe axis X and a movement on the axis Z. In this way, treatments can becarried out along the respective treatment lines 700A to 700F withouttraversing the treatment lines. In this case, by simultaneouslyactivating the nozzle units 720, it is possible to improve detectionenvironments between the treatment lines 700A to 700F, and it ispossible to carry out detection of an amplified product and subsequentanalysis with higher accuracy.

Examples of treatment programs to be stored in the ROM 850 include: (1)a first program by which an RNA is extracted from a cell or virus andamplified to carry out detection of an amplified product; (2) a secondprogram by which a DNA is extracted from a biological sample such asblood and amplified to carry out detection of an amplified product; and(3) a third program by which a plasmid DNA is extracted from a bacteriumor the like. The program to be stored in the ROM may be suitably changeddepending on purposes.

The timer 843 carries out timing according to a program read from theROM 850. Timing is carried out, for example, when isothermalamplification of a target nucleic acid is performed based on a timingclock. By timing, a period of carrying out each step can be accuratelymanaged.

The isothermal control unit 840 has a thermal sensor 870, a temperaturecontroller 872 and a heater 874. The temperature controller 872 detectsa temperature based on a temperature signal from the thermal sensor 870.The thermal sensor 870 is located, for example, adjacent to a well 716holding a freeze-dried reagent for nucleic acid amplification, andtransmits the temperature signal to the temperature controller 872depending on the temperature of a target nucleic acid-containingsolution held in the well 716. The temperature controller 872 controlsenergization of the heater 874 based on the temperature signal from thethermal sensor 870, thereby controlling the temperature of the fluid inthe well 716 to become a predetermined temperature. In this way,isothermal amplification, which requires a constant temperature, or aPCR reaction, which requires temperature change, can be carried out.

The detector 845 has a trigger light source 890, an image sensor 894 forreceiving a light from a POF 740, a detection circuit 896, etc. Byguiding a trigger light from the trigger light source 890 through thePOF 732, the trigger light can be sent to the inside of the well 718 fordetection. For the image sensor 894, for example, image sensors such asCCD and CMOS can be used, and after receiving a light from an imagereproduction optical system 856, an image signal is output to thedetection circuit 896. The detection circuit 896 performs imageprocessing based on a light receiving signal from the image sensor 894to perform detection/determination of nucleic acid.

Next, the action of the nucleic acid detection system of the presentinvention will be described.

The treatment program selected by the user is read from the ROM 850, andbased on the read treatment program, the action of each portion of thenucleic acid detection system 680 is started. By the user, the obtainedspecimen is manually put into the well 702 of each of treatment lines700A to 700F, and by a shielding door or the like (not shown), the firstto sixth treatment lines 700A to 700F are shielded from the outside. Inthe first to sixth treatment lines 700A to 700F, for example, 2 linesare used for extracting a target nucleic acid from a specimen,amplifying it and then detecting it, 2 lines are used for anegative/positive control, and 2 lines are used for producing acalibration curve. Such use may be suitably changed. After shielding ofthe first to sixth treatment lines 700A to 700F is detected, thetreatment of the specimen is started, and the nozzle unit 720 is drivento mix the specimen with the dissolving solution. After stirring of themixture, the magnetic particles are added to the mixture, followed bystirring.

After stirring the mixture to which the magnetic particles have beenadded, the magnetic particles are constrained to obtain magneticparticles to which the target nucleic acid is bound. The obtainedmagnetic particles are mixed with a separating solution for breaking thebond between the magnetic particles and the target nucleic acid. Aftermixing, the target nucleic acid is separated from the magneticparticles, thereby obtaining the target nucleic acid. The obtainedtarget nucleic acid-containing solution is temporarily held in the well714 to be provided to the amplification step. The target nucleicacid-containing solution held in the well 714 is dispensed into the well716 in which a reagent for nucleic acid amplification is held inadvance, and a mineral oil is further dispensed, thereby carrying out anisothermal amplification reaction.

In this regard, as the isothermal amplification reaction, for example,the LAMP (Loop-Mediated Isothermal Amplification) method is used. Aswell known, in the LAMP method, a strand displacement activity enzymeand two primer pairs (inner primer and outer primer) are used to obtainan amplified product. In the LAMP method, there is no need to performthermal denaturation for changing the double strand to the single strandin a template nucleic acid, and therefore, the amplification step can becarried out isothermally. Moreover, when using the PCR method, theamplification cycle of about 5 minutes is repeated at least about 25 to30 times, and in the isothermal nucleic acid amplification method suchas the LAMP method, it is possible to obtain an amplified productsufficient for detection for about 30 minutes. Basic points regardingprimer design are described, for example, in International PublicationWO 2000/28082 pamphlet and International Publication WO 2002/24902pamphlet.

For the isothermal amplification reaction step, in addition to theabove-described LAMP method, for example, the following methods can beemployed:

ICAN (Isothermal and Chimeric primer-initiated Amplification of NucleicAcid) method using a chimeric primer;

RCA (Rolling Cycle Amplification) method in which an amplified productis obtained using open circle probes (OCP), DNA ligase, a primer pairand strand displacement type DNA polymerase;

SDA (Strand Displacement Amplification) method in which an amplifiedproduct is obtained using two primer pairs, a restriction enzyme, astrand displacement activity enzyme and a phosphorothioate analogsubstrate;

IVT (In Vitro Transcription) method;

TRC (Transcription Reverse transcription Concerted amplification) methodin which an RNA is obtained as an amplified product by trimming the RNAusing a reverse transcriptase or the like;

NASBA (Nucleic Acid Sequence-Based Amplification) method in which an RNAis amplified using 3 types of enzymes such as a reverse transcriptaseand an RNA polymerase and a template-specific primer pair; and

SPIA method in which an amplified product is obtained using a primerhaving a chimeric structure of an RNA and a DNA.

Any method, in which a target nucleic acid is amplified at a constanttemperature, can be applied.

Thus, the isothermal amplification reaction step is preferably usedinstead of the PCR method, since there is no need to increase ordecrease the temperature of the nucleic acid solution from roomtemperature to 94° C., 72° C. or 55° C. for amplification of the targetnucleic acid, and therefore there is no need to use an apparatus forincreasing a temperature to a relatively high temperature such as athermal cycler. Further, in the isothermal amplification method, timerequired for obtaining a sufficient amount of amplified product fordetection is about several tens of minutes, and therefore, reduction intime required for extraction to detection of the target nucleic acid canbe expected.

After the extracted target nucleic acid-containing solution is mixedwith the freeze-dried reagent for nucleic acid amplification, ahydrophobic fluid is put into the well 716 by the nozzle unit 720. Asthe hydrophobic fluid, for example, a chain saturated hydrocarbonrepresented by C_(n)H_(2n+2) (n is an integer from 3 to 20), so-calledmineral oil is exemplified, and preferably, liquid paraffin ofC_(n)H_(2n+2) (n is an integer from 16 to 20) is exemplified, and such ahydrophobic fluid is put into the well 716. By the mineral oil, invasionof contaminant from the outside into the well 716 for nucleic acidamplification can be prevented, and in addition, evaporation of a mixedsolution of the target nucleic acid-containing solution and the reagentfor isothermal amplification can be prevented. Further, instead of themineral oil or in combination with the mineral oil, a sealant formed inthe solid state for blocking the opening of the well 716 may be fittedto the opening of the well 716 using the nozzle unit 720.

After amplification of the target nucleic acid, an amplified productionsolution is moved to the well 718 for detection by the nozzle unit 720,and detection of the amplified product is carried out.

Thus, by arranging the well 702 for holding a specimen, a well forholding magnetic particles for target nucleic acid extraction, a wellfor washing and a well for holding a freeze-dried reagent for isothermalamplification in order in a linear fashion, the nozzle unit 720 can bedriven without loss, and further reduction in treatment time can beexpected. Moreover, since the nozzle unit 720 and the pipette chip 730are driven in a linear fashion, mixing of the specimen of anothertreatment line can be prevented, and contamination can be reduced.Furthermore, simultaneously at the 6 treatment lines

Further, the embodiment of the detection apparatus for the targetnucleic acid is not limited to the explanation described above, andvarious changes can be employed. For example, in FIG. 38 describe above,the system has the 6 treatment lines 700A to 700F and the 6 nozzle units720 corresponding to the treatment lines 700A to 700F, but the number ofnozzle units may be one. FIG. 42 is a perspective view schematicallyshowing a target nucleic acid detection apparatus having one nozzleunit. As shown in FIG. 42, a nucleic acid detection apparatus 900 hasone nozzle unit 902. The nozzle unit 902 can be moved along the wellarrangement direction by a first guiding rail 904, and can be movedalong the direction of traversing the first to sixth treatment lines700A to 700F along a second guiding rail 906. By providing such aconstitution of the apparatus, the number of nozzle units may bedecreased, and an apparatus may be provided at low cost.

Further, in the above-described embodiment, the image sensor 894 and thetrigger light source 890 are provided, but a nucleic acid detectionapparatus into which a photomultiplier tube is integrated instead of theimage sensor may also be employed.

Further, a nozzle for dispensing and a detector for target nucleic acidmay be provided independently. As shown in FIG. 43, a nucleic aciddetection apparatus 918 has 6 dispensing nozzles 922 corresponding tofirst to sixth treatment lines 920A to 920F and one nucleic aciddetector 924. The dispensing nozzles 922 are controlled to be moved inthe arrangement direction of wells 926, and the detector 924 is providedabove a well 927 for detection so as to be moved in the direction Straversing the first to sixth treatment lines 920A to 920F. By providingsuch a constitution of the apparatus, efficient treatments can beexpected.

Further, the number of the nucleic acid detector is not limited to one,and a plurality of nucleic acid detectors can be provided correspondingto respective treatment lines as shown in FIG. 44. As shown in FIG. 44,nucleic acid detectors 932 are provided corresponding to first to sixthtreatment lines 930A to 930F. When providing such a constitution of theapparatus, the drive mechanism of the detectors 932 is not required, andtherefore, further improvement of treatment efficiency can be expected.

When the isothermal amplification method is used for the step ofamplifying a target nucleic acid in a nucleic acid amplificationapparatus having a plurality of treatment lines, there is a case wherenucleic acid amplification rates of respective lines are different fromeach other. In this case, reaction temperature may be corrected by atemperature controller. By correcting the temperature of amplificationreaction, the balance between amplification rates of treatment lines canbe expected, and more accurate quantitation of the target nucleic acidcan be expected.

In the explanation above, the pumping opening 730 and the plasticoptical fiber 740 for transmitting an optical image of an amplifiedproduct to the image sensor 894 are provided in the nozzle unit 720, butan optical fiber for transmitting an optical image of an amplifiedproduct to the image sensor may be provided to the outside of thepumping opening. FIG. 45 is a cross sectional view of a unit, in whichan optical fiber for transmitting an optical image of the inside of awell for detection to an image sensor is placed at the outside of apumping opening, taken along a plane approximately parallel to thedrawing direction of an optical fiber. An integrated unit 1000 has anozzle portion 1010 and an optical fiber portion 1020, and the opticalfiber portion 1020 is placed at the outside of the nozzle portion 1010.Thus, the unit in which the optical fiber is placed at the outside ofthe nozzle portion 1010 may also be employed.

Moreover, as shown in FIG. 46, in a nucleic acid detection apparatushaving a plurality of wells for detection, it is also possible toprovide an optical fiber to each of the wells for detection so that anamplified product in each of the wells for detection can be detected,and to selectively use these plurality of optical fibers to detect atarget nucleic acid using a single detector. A nucleic acid detectionapparatus 1050 has: a single image sensor 1052; a switching apparatus1060 for selectively transmitting an optical image of the inside of awell 1054 for detection to the image sensor 1052; a nozzle 1062 fordispensing; treatment lines 1065 in which a plurality of wells forcarrying out extraction to amplification of a target nucleic acid arearranged, etc. It is possible to employ such a constitution of theapparatus, in which an amplified product can be detected from each well1054 for detection by switching of the switching apparatus.

EXAMPLES

Hereinafter, the present invention will be described in more detailbased on Examples, but the present invention is not limited to theExamples.

Example 1 Treatment of AFP-Containing Specimen

[Purpose]

Regarding HAMA and human rheumatoid factor (IgM), contaminant thereofwas treated and removed using Protein G magnetic particles or anti-HumanIgM magnetic particles, and influence of the treatment on values of AFP(α-fetoprotein) was examined.

[Specimens and Apparatuses Used]

ELISA plate to which anti-AFP HYb-2051 is immobilized in advance (F96MAXISORP

NUNC-IMMUNO PLATE (442404, Nunc.))

Serum control (Liquichek Immunoassay Plus Control Level 2, Bio-Rad)

HAMA (Human Anti Mouse Antibody) 1 mg/ml

Dynabeads Protein G (Dynal, Invitrogen) (magnetic particles)

BioMag anti-Human IgM (Bangs Laboratories, Inc.) (magnetic particles)

AFP antigen (Original conc. 4 mg/ml)

AFP-HRP labeling antibody (Original conc. 0.13 mg/ml)

Block Ace Powder (1 pack: 4 g for 100 ml, Cat. No. UK-B80, manufacturedby Snow Brand Milk Products Co., Ltd.)

10×PBS

8 continuous pipettes

Disposable plate

SPECTRAMAX190 (Molecular Devices) & SoftMax Pro 4.8

Nunc-Immuno Wash 8

TMB Peroxidase Substrate & Peroxidase Solution B (H2O2) (KPL: Kirkegaard& Perry Laboratories)

ELISA reaction termination solution (1.0 N H₂SO₄)

Sucker

Kim Towel

[Content of Experiment]

Specimens shown in Sample Nos. 1 to 6 below were prepared, and AFPvalues were measured.

[Calculation of Binding Ability of Magnetic Particles]

For obtaining an AFP value of each specimen, binding ability of magneticparticles was calculated in advance.

The content of each immunoglobulin in serum is shown in Table 1 below.

TABLE 1 IgM IgD IgG1 IgG2 IgG3 IgG4 IgA1 IgA2 IgE Molucular 970 184 146146 165 146 160 160 188 weight (kDa) Serum level 1.5 0.03 9 3 1 0.5 2.00.5 5 × 10⁻⁵ (mg/ml) (%) 8.6 0.2 51.3 17.1 5.7 2.9 11.4 2.9 Reference:The IMMUNE SYSTEM

In 5 μl of serum, about 0.04 to 0.10 mg of IgG is present, and foradsorption to Dynabeads protein G, 100 to 250 μl of beads is required.

Further, in 5 μl of serum, about 7.5 μg of IgM is present, and foradsorption to BioMag anti-Human IgM, 50 μl of beads is required.

[Preparation of Washing Solution]

Block Ace Powder was dissolved in about 90 ml of Milli-Q water. Afterconfirmation of dissolution, the solution was subjected to filling up to100 ml using a 100 ml measuring cylinder. 100 ml of 10×PBS was put intoa 1 L measuring cylinder, Block Ace Powder was added thereto, and themixture was subjected to filling up to 1000 ml by addition of Milli-Qwater.

[Measurement of Absorbance of Each Specimen]

In this experiment, absorbance of each of 6 types of specimens (SampleNos. 1 to 6) was measured. The amount of a reaction specimen on an ELISAplate was 50 μl. As the serum, Liquichek Immunoassay Plus Control Level2 was used. Regarding the labeling antibody AFP-HRP, 1/800 and n=3 wereemployed.

[Sample No. 1]

As a control, PBS (Phosphate buffered salinel) buffer solution was used,and regarding a specimen treated with Dynabeads protein G, a specimentreated with BioMag anti-Human IgM and an untreated specimen, absorbanceof each of them was measured.

[Sample No. 2]

As a serum control, a solution containing 5 μl of serum was used, andregarding a specimen treated with Dynabeads protein G, a specimentreated with BioMag anti-Human IgM and an untreated specimen, absorbanceof each of them was measured.

[Sample No. 3]

A solution containing 5 μl of serum and 10% HAMA was prepared. Usingthis solution, regarding a specimen treated with Dynabeads protein G andan untreated specimen, absorbance of each of them was measured.

[Sample No. 4]

A solution containing 5 μl of serum and 10% rheumatoid factor wasprepared. Using this solution, regarding a specimen treated with BioMaganti-Human IgM and an untreated specimen, absorbance of each of them wasmeasured.

[Sample No. 5]

A solution containing 5 μl of serum, 10% HAMA and 80 ng/ml of AFP wasprepared. Using this solution, regarding a specimen treated withDynabeads protein G and an untreated specimen, absorbance of each ofthem was measured.

[Sample No. 6]

A solution containing 5 μl of serum, 10% rheumatoid factor and 80 ng/mlof AFP was prepared. Using this solution, regarding a specimen treatedwith BioMag anti-Human IgM and an untreated specimen, absorbance of eachof them was measured.

[Results]

Measurement results as shown in Table 2 below were obtained.

TABLE 2 Standard Vriation Sample No. Specimen Treatment Abs450TMBAverage deviation coefficient 1 Control ProteinG 0.0653 0.0642 0.00081790.0127 (PBS 0.0638 buffer solution) 0.0634 Bio Mag 0.0651 0.06670.0017907 0.0268 0.0658 0.0692 No beads 0.0685 0.0651 0.0029033 0.04460.0653 0.0614 2 Serum control ProteinG 0.2777 0.2722 0.0041721 0.01530.2713 0.2676 Bio Mag 0.2841 0.2872 0.0030232 0.0105 0.2862 0.2913 Nobeads 0.2959 0.3002 0.0076613 0.0255 0.2938 0.3110 3 Serum + HAMAProteinG 0.2631 0.2543 0.006287 0.0247 0.2510 0.2488 No Beads 1.24721.2696 0.0182491 0.0144 1.2698 1.2919 4 Serum + Bio Mag 0.7160 0.69170.0211292 0.0305 rheumatoid 0.6947 factor 0.6645 No Beads 0.7215 0.72310.0016083 0.0022 0.7225 0.7253 5 Serum + ProteinG 0.8900 0.86970.0143781 0.0165 HAMA + 0.8594 AFP 0.8596 No Beads 1.4009 1.43920.0273761 0.0190 1.4537 1.4631 6 Serum + Bio Mag 1.3443 1.3308 0.01280340.0096 rheumatoid 1.3345 factor + 1.3136 AFP No Beads 1.3512 1.36750.0117284 0.0086 1.3732 1.3782[Calculation of AFP]

AFP values were calculated based on values of Abs450TMB shown in Table 2above. For calculation of AFP values, among the following mathematicalformulae obtained from FIGS. 47 and 48:y=0.0065x+0.2088  Formula (1)y=0.0068x+0.0554  Formula (2),Formula (1) was used.

The calculated AFP values are shown in Table 3 below.

TABLE 3 Sample Beads AFP No. Specimen Treatment treatment value [ng/ml]1 Control ProteinG Done 1.29 (PBS Bio Mag Done 1.66 buffer solution) Nobeads Not done 1.43 2 Serum control ProteinG Done 9.75 Bio Mag Done12.06 No beads Not done 14.06 3 Serum + HAMA ProteinG Done 7.00 No BeadsNot done 163.20 4 Serum + Bio Mag Done 74.30 rheumatoid No Beads Notdone 79.12 factor 5 Serum + ProteinG Done 101.68 HAMA + No Beads Notdone 189.29 AFP 6 Serum + Bio Mag Done ※Substract 66.20 rheumatoid NoBeads Not done ※Substract 67.02 factor + AFP ※shows difference fromSample 4.[Consideration Regarding Experiment Results]

As shown by the AFP value in the case of no treatment (“no beads”treatment) of Sample No. 3, the AFP value was increased due to thepresence of HAMA, and therefore, it is considered that there is apossibility that HAMA is a false-positive factor.

In order to confirm this consideration, HAMA was treated with Dynabeadsprotein G. As a result, the AFP value in the case of treatment of SampleNo. 3 with Dynabeads protein G was 7.00, and the AFP value was decreasedto a level similar to that of the AFP value shown for Sample No. 2.

Therefore, it was confirmed that HAMA is a factor causing falsepositive.

Regarding the AFP values of Sample No. 4, the AFP value in the case oftreatment with BioMag was almost the same as the AFP value in the caseof no treatment (“no beads” treatment).

CONCLUSION

By treating and removing the contaminant contained in the specimen usingProtein G Dynabeads, IgM, which is thought to be the cause for falsepositive, can be removed from the specimen. Therefore, the amount of AFPexisting in the specimen can be more accurately measured.

EXPLANATIONS OF LETTERS OR NUMERALS

-   10 pipette chip-   12 well-   20 column-containing pipette chip-   30 membrane-containing pipette chip-   40 gel-containing pipette chip-   50 pipette chip (tubular chip)-   70 antigen separation/immobilization tube-   72 spacer beads-   100 assay system-   102 central processing unit-   106 chip mount controller-   108 magnetic field controller-   112 pumping controller-   130 magnet-   140 pump-   146 pressure sensor-   150 assay system-   151 magnet-   152 dispensing apparatus-   153 heat block-   154 detection apparatus-   162 nozzle-   164 pipette chip-   170 PMT-   171 light source-   180 cartridge-   181 base panel-   182 holding portion-   260 assay system-   262 system body-   264 cartridge

The invention claimed is:
 1. A biologically-relevant substance assay device, comprising: (a) a specimen holding portion in which a specimen is held; (b) a first holding portion in which magnetic particles for trapping a biologically-relevant substance from the specimen are held; (c) a second holding portion in which a reagent for detecting the biologically-relevant substance is held; and (d) a dispensing mechanism for dispensing the specimen into the specimen holding portion; wherein the specimen holding portion, the first holding portion, and the second holding portion are arranged in line and in the order according to the content of the treatment of the specimen and the biologically-relevant substance, and each of the first holding portion and the second holding portion includes a plurality of wells, each well being separated from adjacent wells by a well wall, wherein the device further comprises a dispensing position controller configured for controlling the position of the dispensing mechanism, a magnet configured for constraining the magnetic particles in a nozzle of the dispensing mechanism, and a magnetic field controller configured for managing the placement of the magnet to control the strength of the magnetic field provided to the nozzle of the dispensing mechanism, the dispensing position controller moves the dispensing mechanism in a perpendicular direction with respect to the line and in a horizontal direction parallel to the line, and the magnetic field controller moves the magnet in the horizontal direction parallel to the line, and wherein a tip portion of the nozzle has a pumping opening, at least one optical fiber configured for sending a trigger light and a lens for receiving light from the biologically-relevant substance.
 2. The device according to claim 1, wherein the magnetic particles are held in a dispensing chip.
 3. The device according to claim 1, further having a mechanism for sliding upper portions of the holding portions.
 4. The device according to claim 1, wherein the specimen holding portion, the first holding portion and the second holding portion are arranged in an approximate straight line.
 5. The device according to claim 1, wherein the reagent for detecting is freeze-dried in advance.
 6. The device according to claim 1, wherein the first and second holding portions are formed into a cartridge.
 7. The device according to claim 6, wherein the specimen holding portion, the first holding portion, and the second holding portion are integrated into the cartridge.
 8. The device according to claim 1, wherein the biologically relevant substance is a nucleic acid.
 9. The device according to claim 8, wherein the reagent for detecting comprises a reagent for nucleic acid amplification using a PCR method or an isothermal amplification method and a reagent for detecting an amplified product.
 10. The device according to claim 1, wherein the reagent for detecting is a freeze-dried reagent, and the device further includes a well containing the freeze dried reagent, and the biologically-relevant substance is detected by using the freeze-dried reagent.
 11. The device according to claim 1, further comprising one or more of treatment lines including the specimen holding portion, the first holding portion, and the second holding portion.
 12. The device according to claim 11, further comprising one or more of nozzle units linearly moved along the one or more of treatment lines.
 13. The device according to claim 1, further comprising (e) a mechanism selected from the group consisting of: a mechanism for removing or putting lids for covering the respective holding portions; a mechanism for receiving a light from the biologically-relevant substance irradiated with the irradiating light to detect the biologically relevant substance; and a mechanism in which the mechanisms are combined.
 14. The device according to claim 13, wherein the mechanisms in (d) and the mechanism in (e) or a mechanism in which the mechanisms in (d) and the mechanism in (e) are combined are installed in a single nozzle.
 15. The device according to claim 1, wherein the lens is placed in the pumping opening.
 16. The device according to claim 1, wherein a plurality of the at least one optical fiber are arranged around the pumping opening.
 17. The device according to claim 1, wherein the pumping opening and the lens are disposed in the central portion of the tip portion of the nozzle.
 18. A method for assaying a nucleic acid, wherein the nucleic acid is extracted and detected using the device according to claim
 1. 